David G. Reid

Rapid reversal of hepatic steatosis, and reduction of muscle triglyceride, by rosiglitazone: MRI/S studies in Zucker fatty rats.

Aim:  This study aimed to chart the time course and durability of the effects of rosiglitazone, a potent thiazolidinedione‐based peroxisome proliferator‐activated receptor γ agonist, on hepatic steatosis and intramyocellular lipid in an animal model of obesity, the Zucker Fatty (ZF) rat.

The conformation of an inhibitor bound to the gastric proton pump

Substituted imidazo[1,2‐a]pyridines are pharmaceutically important small molecule inhibitors of the gastric H+/K+‐ATPase, the membrane‐bound therapeutic target for peptic ulcer disease. A non‐perturbing analytical technique, rotational resonance NMR spectroscopy, was used to measure a precise (to ±0.2 Å) distance between atomic sites in a substituted imidazo[1,2‐a]pyridine, TMPIP, bound to H+/K+‐ATPase at its high‐affinity site in the intact, native membrane. The structural analysis of the enzyme–inhibitor complex revealed that the flexible moiety of TMPIP adopts a ‘syn‐type’ conformation at its site of action. Hence, the conformation of an inhibitor has been resolved directly under near‐physiological conditions, providing a sound experimental basis for rational design of many active compounds of pharmaceutical interest. Chemically restraining the flexible moiety of compounds like TMPIP in the syn‐type binding conformation was found to increase activity by over 2 orders of magnitude. Such information is normally only available after extensive synthesis of related compounds and multiple screening approaches.

Metabolomic study of the LDL receptor null mouse fed a high-fat diet reveals profound perturbations in choline metabolism that are shared with ApoE null mice

Failure to express or expression of dysfunctional low-density lipoprotein receptors (LDLR) causes familial hypercholesterolemia in humans, a disease characterized by elevated blood cholesterol concentrations, xanthomas, and coronary heart disease, providing compelling evidence that high blood cholesterol concentrations cause atherosclerosis. In this study, we used (1)H nuclear magnetic resonance spectroscopy to examine the metabolic profiles of plasma and urine from the LDLR knockout mice. Consistent with previous studies, these mice developed hypercholesterolemia and atherosclerosis when fed a high-fat/cholesterol/cholate-containing diet. In addition, multivariate statistical analysis of the metabolomic data highlighted significant differences in tricarboxylic acid cycle and fatty acid metabolism, as a result of high-fat/cholesterol diet feeding. Our metabolomic study also demonstrates that the effect of high-fat/cholesterol/cholate diet, LDLR gene deficiency, and the diet-genotype interaction caused a significant perturbation in choline metabolism, notably the choline oxidation pathway. Specifically, the loss in the LDLR caused a marked reduction in the urinary excretion of betaine and dimethylglycine, especially when the mice are fed a high-fat/cholesterol/cholate diet. Furthermore, as we demonstrate that these metabolic changes are comparable with those detected in ApoE knockout mice fed the same high-fat/cholesterol/cholate diet they may be useful for monitoring the onset of atherosclerosis across animal models.

Conformation of the cytoplasmic domain of phospholamban by NMR and CD

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.

Short term arterial remodelling in the aortae of cholesterol fed New Zealand white rabbits shown in vivo by high-resolution magnetic resonance imaging - implications for human pathology.

High-resolution, non-invasive imaging methods are required to monitor progression and regression of atherosclerotic plaques. We investigated the use of MRI to measure changes in plaque volume and vessel remodelling during progression and regression of atherosclerosis in New Zealand White rabbits. Atherosclerotic lesions were induced in the abdominal aorta by balloon injury and cholesterol feeding. MR images (2D) of the abdominal aorta were acquired with cardiac and respiratory gating using a fast spin echo sequence with and without fat-suppression. In an initial study on rabbits treated for 30 weeks we imaged the aortae with a spatial resolution of 250×250 micrometers with a slice thickness of 2 mm and achieved a close correlation between MRI-derived measurements and those made on perfusion pressure-fixed histological sections (r1 = 0.83, slope p1 < 0.01). We subsequently imaged 18 rabbits before and periodically during 12 weeks of cholesterol feeding (progression) followed by 12 weeks on normal diet (regression). Aortic wall (atherosclerotic lesion) volume increased significantly during progression and decreased during regression. In contrast, lumen volume increased during progression and did not change during regression. In conclusion, this study confirms that non-invasive, high-resolution MRI can be used to monitor progression and regression of atherosclerosis, each within 3 months and shows, for the first time in a short-term model, that positive remodelling occurs early during progression and persists through regression of atherosclerotic lesions.

A solid-state NMR study of the phospholamban transmembrane domain: local structure and interactions with Ca2+-ATPase

Abstract The structure and dynamics of a double 13C-labelled 24-residue synthetic peptide ([13C2]CAPLB29–52), corresponding to the membrane-spanning sequence of phospholamban (PLB), were examined using 13C cross-polarisation magic-angle spinning (CP-MAS) NMR spectroscopy. CP-MAS spectra of [13C2]CAPLB29–52 reconstituted into unsaturated lipid membranes indicated that the peptide was mobile at temperatures down to −50°C. The NMR spectra showed that peptide motion became constrained in the presence of the SERCA1 isoform of Ca2+-ATPase, and chemical cross-linking experiments indicated that [13C2]CAPLB29–52 and Ca2+-ATPase came into close contact with one another. These results together suggested that the peptide and the 110-kDa calcium pump were interacting in the membrane. Rotational resonance CP-MAS 13C–13C distance measurements on [13C2]CAPLB29–52 reconstituted into lipid bilayers confirmed that the sequence spanning Phe-32 and Ala-36 was α-helical, and that this structure was not disrupted by interaction with Ca2+-ATPase. These results support the finding that the transmembrane domain of PLB is partially responsible for regulation of Ca2+ transport through interactions with cardiac muscle Ca2+-ATPase in the lipid bilayer, and also demonstrate the feasibility of performing structural measurements on PLB peptides when bound to their physiological target.

Validation of MRI Measurement of Cardiac Output in the Dog: The Effects of Dobutamine and Minoxidil

The use of magnetic resonance imaging (MRI) for the measurement of cardiac output parameters in anesthetized adult male beagle dogs has been validated against a widely accepted thermodilution method. Using a multislice cine gradient echo MRI method to acquire images of the entire heart, left ventricular lumen volumes were measured at systole and diastole in seven animals. Cardiac output correlated well (R 2 = 0.88) with thermodilution measurements made in a parallel manner, both before and during acute stimulation with the inotrope dobutamine. In a chronic study of changes in cardiac morphology and function brought about by the antihypertensive minoxidil, MRI reliably detected the expected increases in stroke volume (28%) and cardiac output (58%) resulting from neural reaction to decreased blood pressure. Left ventricular lumen enlarged as well in response to fluid retention and plasma volume increase. Two in four minoxidil-treated animals also developed clear MRI-visible pericardial effusion.

Molecular Dynamics of Poly(Lactide-Co-Glycolide) Controlled Pharmaceutical Release Polymers: Preliminary Solid State NMR

Abstract Variable temperature 13C cross polarization magic angle spinning (CP-MAS) and 1H T 1 and T 1p studies have been initiated on microspheres of the poly(lactide-co-glycolide) (PLGA) materials widely studied as controlled release pharmaceutical formulation excipients. Temperature dependence of lineshape and T 1p suggest that around the broad glass transition at about 50°C slow polymer backbone motions (on the 10 to 100 μs time scale) become significant; modelling of these is in progress.

MRI quantification in vivo of corticosteroid induced thymus involution in mice: Correlation with ex vivo measurements

Thymus involution is a useful marker of transactivation-mediated side effects in preclinical therapeutic index testing of new anti-inflammatory glucocorticosteroids, and is usually measured post mortem. We have validated the use of MRI for non-invasive in vivo measurement of mouse thymus involution induced by dexamethasone (DEX). Tl-weighted spin echo 7 T images provided satisfactory contrast between thymus and surrounding connective tissue and fat. Increasing doses of DEX caused thymus involution, reflected in MRI volume (87+/-14, 33+/-10, 28+/-6, 16+/-7 microl in dosage groups of Cremophor vehicle, 1, 10 and 30 mg/kg subcutaneous respectively, n=6/group, mean+/-standard deviation) and post mortem wet weight (64+/-12, 33+/-6, 25+/-9, 23+/-8 mg). Correlation between MRI volumes and wet weights was very good (r=0.842). Measuring pre-dose MRI volumes and then assessing DEX effects as post-dose change from baseline produced no statistical advantage relative to considering post-dose MRI thymus volume alone, probably due to variability in pre-dose baseline values compounding post-dose variability. Smaller group sizes were sufficient to achieve a given statistical power using MRI post-dose volume than using wet weight, suggesting a role for MRI in differentiating the effects of compounds which produce similar effects, or in contexts where the use of large groups of animals is impractical or ethically unacceptable.

Synthesis and analysis of the enantiomers of calmidazolium, and a 1H NMR demonstration of a chiral interaction with calmodulin

Calmidazolium [R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4- dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride] is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole [1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4 dichlorophenyl))-ethyl imidazole], prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the 1H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d5 calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the 1H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation.