The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

1. Potency orders were determined for a series of agonists and antagonists on the calcitonin gene-related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. 2. AC187 had a PIC50 Of 6.8 ± 0.10, making it 14 fold less potent as an antagonist than CGRP8-37 (PIC50, 7.95 ± 0.14). Amyline8-37 was equipotent to AC187 (PIC50, 6.6 ± 0.16) and CGRP19-37 was a fold less potent than either (pIC50 6.1 ± 0.24). 3. [Ala11]-CGRP8-37 was 6 fold less potent than CGRP8-37, (pIC50 7.13 ± 0.14), whereas [Ala18] CGRP8-37 was approximately equipotent to CGRP8-37 (pIC50, 7.52 ± 0.15). However, [Ala11,Ala18]- CGRP8-37 was over 300 fold less potent than CGRP8-37 (pIC50, 5.30 ± 0.04). 4. [Tyr0]-CGRP28-37, amylin19-37 and adrenomedullin22-52 were inactive as antagonists at concentrations of up to 1 μM. 5. Biotinyl-human α-CGRP was 150 fold less potent than human α-CGRP itself (EC50 values of 48 ± 17 nM and 0.31 ± 0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)(2'7)]-CGRP was inactive as an agonist. 6. These results confirm a role for Arg11 in maintaining the high affinity binding of CGRP8-37. Arg18 is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.

Visual field and structural alterations in age-related macular degeneration

The thesis investigated progression of the central 10° visual field with structural changes at the macula in a cross-section of patients with varying degrees of agerelated macular degeneration (AMD). The relationships between structure and function were investigated for both standard and short-wavelength automated perimetry (SWAP). Factors known to influence the measure of visual field progression were considered, including the accuracy of the refractive correction on SWAP thresholds and the learning effect. Techniques of assessing the structure to function relationships between fundus images and the visual field were developed with computer programming and evaluated for repeatability. Drusen quantification of fundus photographs and retro-mode scanning laser ophthalmoscopic images was performed. Visual field progression was related to structural changes derived from both manual and automated methods. Principal Findings: • Visual field sensitivity declined with advancing stage of AMD. SWAP showed greater sensitivity to progressive changes than standard perimetry. • Defects were confined to the central 5°. SWAP defects occurred at similar locations but were deeper and wider than corresponding standard perimetry defects. • The central field became less uniform as severity of AMD increased. SWAP visual field indices of focal loss were of more importance when detecting early change in AMD, than indices of diffuse loss. • The decline in visual field sensitivity over stage of severity of AMD was not uniform, whereas a linear relationship was found between the automated measure of drusen area and visual field parameters. • Perimetry exhibited a stronger relationship with drusen area than other measures of visual function. • Overcorrection of the refraction for the working distance in SWAP should be avoided in subjects with insufficient accommodative facility. • The perimetric learning effect in the 10° field did not differ significantly between normal subjects and AMD patients. • Subretinal deposits appeared more numerous in retro-mode imaging than in fundus photography.

Synthesis and structural examination of 4-azaindoles

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Visual field and structural correlates of progression in age-related macular degeneration

Presentation Purpose:To examine the correlation of central visual field loss and progression of structural changes in the macular area in age-related macular degeneration (AMD). Methods:Central 10° standard and short-wavelength automated perimetry (SWAP) visual fields were acquired in 39 eyes of 24 patients with AMD using a Humphrey Field Analyzer. Stereoscopic fundus photographs were graded1 by two independent observers and the stage of disease determined2. Custom software mapped perimetric data onto fundus images in order to relate structural changes to functional loss. Results:Mean deviation (MD) in standard perimetry changed from 0.04 dB at stage 1 to -12.39 dB at stage 4 (r2=0.48, p<0.001). The group mean SWAP MD was -5.26 dB at stage 1 and increased to -17.08 dB at stage 4 (r2=0.53, p<0.001). Pattern standard deviation (PSD) also increased with advancing stage in standard perimetry; 1.32 dB to 8.67 dB at stage 1 and 4, respectively (r2=0.54, p<0.001). In SWAP, PSD increased from 2.86 dB to 5.63 dB at stage 1 and stage 4 (r2=0.43, p<0.001). Defect frequency was greater in SWAP than standard perimetry. Early stage defects occurred with the greatest frequency at eccentricities of 3.2° and 5.1° in standard perimetry and at 4.2° in SWAP. Late stage defects were most frequent at 1° eccentricity in standard perimetry and at 1° and 9° in SWAP. MD declined with increasing affected retinal area over the central 3000µm; by 0.20 dB (r2=0.67, p<0.001) and 0.18 dB (r2=0.49, p<0.001) per % increase in defect area for standard perimetry and SWAP respectively. 41% of defects were associated with structural changes on the retina in standard perimetry and 43% in SWAP. Conclusions:Sensitivity decreased with advancing stage of AMD, with a greater effect demonstrated in SWAP compared to standard perimetry. The central field became less uniform as stage increased. SWAP defects occurred at similar locations but were deeper and wider than corresponding defects in standard perimetry. Central loss in SWAP is a sensitive marker of functional progression in AMD.1. Bird et al. (1995) Surv Ophthalmol 39:367-3742. van Leeuwen et al. (2003) Arch Ophthalmol 121:519-526

Purchasing power parity and structural instability in the US/UK exchange rate

The aim of this study is to determine if nonlinearities have affected purchasing power parity (PPP) since 1885. Also using recent advances in the econometrics of structural change we segment the sample space according to the identified breaks and look at whether the PPP condition holds in each sub-sample and whether this involves linear or non-linear adjustment. Our results suggest that during some sub-periods, PPP holds, although whether it holds or not and whether the adjustment is linear or non-linear, depends primarily on the type of exchange rate regime in operation at any point in time.

Quality of life, stress and mental health in parents of children with parentally diagnosed food allergy compared to medically diagnosed and healthy controls

Background. Food allergy is related to poorer quality of life (QoL) and mental health of caregivers. Many parents diagnose food allergy in their child without seeking medical care and there is limited research on this group. This study investigated parental QoL and mental health in parents of children with parent-diagnosed food allergy (PA), medically diagnosed food allergy (MA), and a control group with no allergy (NA). Methods. One hundred and fifty parents from a general population completed validated measures of QoL, anxiety, depression, and stress. Results. Parents of children with food allergy (PA or MA) reported higher stress, anxiety, and depression than the control group (all ). Parents of children with MA reported poorer food allergy related QoL compared to parents of children with PA (); parents of children with PA reported poorer general QoL compared to parents of children with MA (). Conclusion. Parents of children with food allergy have significantly poorer mental health compared to healthy controls, irrespective of whether food allergy is medically diagnosed or not. It is important to encourage parents to have their child medically tested for food allergy and to recognise and refer for psychological support where needed.

Risk and structural instability in US house prices

This paper employs a Component GARCH in Mean model to show that house prices across a number of major US cities between 1987 and 2009 have displayed asset market properties in terms of both risk-return relationships and asymmetric adjustment to shocks. In addition, tests for structural breaks in the mean and variance indicate structural instability across the data range. Multiple breaks are identified across all cities, particularly for the early 1990s and during the post-2007 financial crisis as housing has become an increasingly risky asset. Estimating the models over the individual sub-samples suggests that over the last 20 years the financial sector has increasingly failed to account for the levels of risk associated with real estate markets. This result has possible implications for the way in which financial institutions should be regulated in the future.

Deindustrialisation and structural change during the post-Communist transition

The aim of this paper is to model the evolution of employment structure in post-communist economies in the broader context of deindustrialisation. The paper builds on the model of structural change developed by Rowthorn and Wells (1987). We show that the starting point of high industry sector share in total employment and its direct fall when productivity of sectors changes in favour of services can be explained in terms of this framework. Moreover, the model can also describe the phenomenon of a further expansion of the agriculture, observed in countries classified as "less consistent" in the reforms implementation. Hence, we distinguish two development paths, the efficient one, called "horizontal", and the inefficient one called "vertical". We illustrate it with empirical data, using alternative measures of structural change and patterns of structural evolutions during transition. Finally, we discuss the link between the EBRD indicators of reforms and structural change. We show that the "quality" of reforms, not the initial GDP level determines a country's development path.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Aminoglutethimide-induced leucopenia in a mouse model:effects of metabolic and structural determinates

A model of human leucopenia has been developed further in the female mouse. Following daily administration to female mice of 50 mg/kg of the aromatase inhibitor aminoglutethimide, significant falls in platelet and white cell counts occurred after 2 and 3 weeks. At week 4, drug dosage was stopped and the cell counts recovered at the end of that week, although on rechallenge at the beginning of week 5, both platelet and white cell counts fell rapidly. Administration to the mice of structural analogues of aminoglutethimide, such as WSP-3, glutethimide and 4-nitroglutethimide, showed no reductions in platelet and white cell counts. The haemotoxicity of aminoglutethimide over 21 days was unaffected by the presence of either the P-450 inhibitor SKF-525A or the hepatic P-450 inducer phenobarbitone. However, the co-administration of cimetidine abolished the haemotoxicity of aminoglutethimide in terms of platelet and white cell levels. In in vitro studies, both aminoglutethimide and WSP-3 were oxidised to cytotoxic species, although aminoglutethimide was significantly more cytotoxic than WSP-3. The NADPH-dependent covalent binding of 14C aminoglutethimide to mouse microsomes in vitro was significantly reduced by the presence of cimetidine. The activation of the compound to reactive species in vitro, the inhibitory effects of cimetidine in vivo and in vitro, as well as the rapid fall in the in vivo white cell count on rechallenge with aminoglutethimide suggest that this model illustrates a form of leucopenia which may be related to hapten formation and subsequent immune-mediated platelet and white cell lysis. © 2003 Elsevier B.V. All rights reserved.

Optical and structural ocular changes during incipient presbyopia

The primary aim of this thesis was to investigate the in vivo ocular morphological and contractile changes occurring within the accommodative apparatus prior to the onset of presbyopia, with particular reference to ciliary muscle changes with age and the origin of a myopic shift in refraction during incipient presbyopia. Commissioned semi-automated software proved capable of extracting accurate and repeatable measurements from crystalline lens and ciliary muscle Anterior Segment Optical Coherence Tomography (AS-OCT) images and reduced the subjectivity of AS-OCT image analysis. AS-OCT was utilised to document longitudinal changes in ciliary muscle morphology within an incipient presbyopic population (n=51). A significant antero-inwards shift of ciliary muscle mass was observed after 2.5 years. Furthermore, in a subgroup study (n=20), an accommodative antero-inwards movement of ciliary muscle mass was evident. After 2.5 years, the centripetal response of the ciliary muscle significantly attenuated during accommodation, whereas the antero-posterior mobility of the ciliary muscle remained invariant. Additionally, longitudinal measurement of ocular biometry revealed a significant increase in crystalline lens thickness and a corresponding decrease in anterior chamber depth after 2.5 years (n=51). Lenticular changes appear to be determinant of changes in refraction during incipient presbyopia. During accommodation, a significant increase in crystalline lens thickness and axial length was observed, whereas anterior chamber depth decreased (n=20). The change in ocular biometry per dioptre of accommodation exerted remained invariant after 2.5 years. Cross-sectional ocular biometric data were collected to quantify accommodative axial length changes from early adulthood to advanced presbyopia (n=72). Accommodative axial length elongation significantly attenuated during presbyopia, which was consistent with a significant increase in ocular rigidity during presbyopia. The studies presented in this thesis support the Helmholtz theory of accommodation and despite the reduction in centripetal ciliary muscle contractile response with age, primarily implicate lenticular changes in the development of presbyopia.

Dissociable brain structural changes associated with predisposition, resilience, and disease expression in bipolar disorder

Genetic factors are important in the etiology of bipolar disorder (BD). However, first-degree relatives of BD patients are at risk for a number of psychiatric conditions, most commonly major depressive disorder (MDD), although the majority remain well. The purpose of the present study was to identify potential brain structural correlates for risk and resilience to mood disorders in patients with BD, type I (BD-I) and their relatives. Structural magnetic resonance imaging scans were acquired from 30 patients with BD-I, 50 of their firstdegree relatives (28 had no Axis I disorder, while 14 had MDD) and 52 controls. We used voxel-based morphometry, implemented in SPM5 to identify group differences in regional gray matter volume. From the identified clusters, potential differences were further examined based on diagnostic status (BD-I patients, MDD relatives, healthy relatives, controls). Whole-brain voxel-based analysis identified group differences in the left hemisphere in the insula, cerebellum, and substantia nigra. Increased left insula volume was associated with genetic preposition to BD-I independent of clinical phenotype. In contrast, increased left substantia nigra volume was observed in those with the clinical phenotype of BD-I. Changes uniquely associated with the absence of a clinical diagnosis in BD relatives were observed in the left cerebellum. Our data suggest that in BD, genetic and phenotype-related influences on brain structure are dissociable; if replicated, these findings may help with early identification of high-risk individuals who are more likely to transition to syndromal states. Copyright © 2009 Society for Neuroscience.

Structural properties of a family of hydrogen-bonded co-crystals formed between gemfibrozil and hydroxy derivatives of t-butylamine, determined directly from powder X-ray diffraction data

We report the formation and structural properties of co-crystals containing gemfibrozil and hydroxy derivatives of t-butylamine H2NC(CH3)3-n(CH2OH)n, with n=0, 1, 2 and 3. In each case, a 1:1 co-crystal is formed, with transfer of a proton from the carboxylic acid group of gemfibrozil to the amino group of the t-butylamine derivative. All of the co-crystal materials prepared are polycrystalline powders, and do not contain single crystals of suitable size and/or quality for single crystal X-ray diffraction studies. Structure determination of these materials has been carried out directly from powder X-ray diffraction data, using the direct-space Genetic Algorithm technique for structure solution followed by Rietveld refinement. The structural chemistry of this series of co-crystal materials reveals well-defined structural trends within the first three members of the family (n=0, 1, 2), but significantly contrasting structural properties for the member with n=3. © 2007 Elsevier Inc. All rights reserved.

Affect networks:a structural analysis of the relationship between work ties and job-related affect

The relationship between organizational networks and employees' affect was examined in 2 organizations. In Study 1, social network analysis of work ties and job-related affect for 259 employees showed that affect converged within work interaction groups. Similarity of affect between employees depended on the presence of work ties and structural equivalence. Affect was also related to the size and density of employees' work networks. Study 2 used a 10-week diary study of 31 employees to examine a merger of 2 organizational divisions and found that negative changes in employees' affect were related to having fewer cross-divisional ties and to experiencing greater reductions in network density. The findings suggest that affect permeates through and is shaped by organizational networks.