Kevin Hallock

MSI-78, an Analogue of the Magainin Antimicrobial Peptides, Disrupts Lipid Bilayer Structure via Positive Curvature Strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.

Membrane Composition Determines Pardaxin's Mechanism of Lipid Bilayer Disruption

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptides C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptides ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.

NMR structure of pardaxin, a pore-forming antimicrobial peptide, in lipopolysaccharide Micelles: Mechanism of outer membrane permeabilization

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is an important element against permeability of bactericidal agents, including antimicrobial peptides. However, structural determinants of antimicrobial peptides for LPS recognition are not clearly understood. Pardaxins (Pa1, Pa2, Pa3, and Pa4) are a group of pore-forming bactericidal peptides found in the mucous glands of sole fishes. Despite having a low net positive charge, pardaxins contain a broad spectrum of antibacterial activities. To elucidate the structural basis of LPS interactions of pardaxins, herein, we report the first three-dimensional structure of Pa4 bound to LPS micelles. The binding kinetics of Pa4 with LPS is estimated using [15N-Leu-19] relaxation dispersion NMR experiments. LPS/Pa4 interactions are further characterized by a number of biophysical methods, including isothermal titration calorimetry, 31P NMR, saturation transfer difference NMR, dynamic light scattering, and IR spectroscopy. In the LPS-Pa4 complex, Pa4 adopts a unique helix-turn-helix conformation resembling a “horseshoe.” Interestingly, the LPS-bound structure of Pa4 shows striking differences with the structures determined in lipid micelles or organic solvents. Saturation transfer difference NMR identifies residues of Pa4 that are intimately associated with LPS micelles. Collectively, our results provide mechanistic insights into the outer membrane permeabilization by pardaxin.

An Innovative Procedure Using a Sublimable Solid to Align Lipid Bilayers for Solid-State NMR Studies

Uniaxially aligned phospholipid bilayers are often used as model membranes to obtain structural details of membrane-associated molecules, such as peptides, proteins, drugs, and cholesterol. Well-aligned bilayer samples can be difficult to prepare and no universal procedure has been reported that orients all combinations of membrane-embedded components. In this study, a new method for producing mechanically aligned phospholipid bilayer samples using naphthalene, a sublimable solid, was developed. Using (31)P-NMR spectroscopy, comparison of a conventional method of preparing mechanically aligned samples with the new naphthalene procedure found that the use of naphthalene significantly enhanced the alignment of 3:1 1-palmitoyl-2-oleoyl-phosphatidylethanolamine to 1-palmitoyl-2-oleoyl-phosphatidylglycerol. The utility of the naphthalene procedure is also demonstrated on bilayers of many different compositions, including bilayers containing peptides such as pardaxin and gramicidin. These results show that the naphthalene procedure is a generally applicable method for producing mechanically aligned samples for use in NMR spectroscopy. The increase in bilayer alignment implies that this procedure will improve the sensitivity of solid-state NMR experiments, in particular those techniques that detect low-sensitivity nuclei, such as 15N and 13C.

Diffusion tensor spectroscopy and imaging of the arcuate fasciculus

The arcuate fasciculus (AF) is a fiber pathway in the human brain relevant for language processes and has recently been characterized by means of diffusion tensor tractography. The observations made concerning the left and right hemisphere AF include a characterization of the trajectories and quantification of physical properties such as fractional anisotropy, DTI-based fiber density and volume. However, these observations were based on the diffusion of water, which is not particular to either the intra- or extra-axonal compartments, and thus its usefulness for tissue characterization is limited. If the diffusion properties and in turn the geometric properties of only one tissue compartment can be isolated and characterized, a better microstructural characterization of AF is possible. In this study, water-based diffusion tensor probabilistic mapping was first implemented to segment the AF. Subsequently, diffusion tensor spectroscopic measurements of N-acetyl aspartate (NAA) were performed to measure the intra-axonal specific diffusion in left and right AF. Diffusion properties of NAA, which solely reflect the intra-axonal space, indicated possible leftward asymmetry in axonal diameter, where those of water, which are not compartment-specific, showed laterality to a lesser extent.

In vivo detection of vulnerable atherosclerotic plaque by MRI in a rabbit model

Background—The ability to identify atherosclerotic plaques with a high risk for sudden disruption before stroke or myocardial infarction would be of great utility. We used a rabbit model of controlled atherothrombosis to test whether in vivo MRI can noninvasively distinguish between plaques that disrupt after pharmacological triggering (vulnerable) and those that do not (stable). Methods and Results—Atherosclerosis was induced in male New Zealand White (n=17) rabbits by cholesterol diet and endothelial denudation of the abdominal aorta. After baseline (pretrigger) MRI with and without gadolinium contrast, the rabbits underwent 2 pharmacological triggerings to induce atherothrombosis, followed by another MRI 48 hours later (post-triggering). Atherosclerosis was identified by the pretriggered images in all rabbits, and thrombosis was identified in 9 of 17 animals (53%) by post-trigger MRI. After the animals were euthanized, 95 plaques were analyzed; 28 (29.5%) had thrombi (vulnerable) and 67 did not (stable) (70.5%). Pretriggered MRI revealed comparable stenosis in stable and vulnerable plaques, but vulnerable plaques had a larger plaque area (4.8±1.6 versus 3.0±1.0 mm2; P=0.01), vessel area (9.2±3.0 versus. 15.8±4.9 mm2; P=0.01), and higher remodeling ratio (1.16±0.2 versus 0.93±0.2; P=0.01) compared with stable plaques. Furthermore, vulnerable plaques more frequently exhibited (1) positive remodeling (67.8% versus 22.3%; P=0.01), in which the plaque is hidden within the vessel wall instead of occluding the lumen; and (2) enhanced gadolinium uptake (78.6% versus 20.9%; P=0.01) associated with histological findings of neovascularization, inflammation, and tissue necrosis. Conclusions—We demonstrate that in vivo MRI at 3.0 T detects features of vulnerable plaques in an animal model of controlled atherothrombosis. These findings suggest that MRI may be used as a noninvasive modality for localization of plaques that are prone to disruption.

In vivo Detection of Vulnerable Atherosclerotic Plaque by Magnetic Resonance Imaging in a Rabbit Model

Background—The ability to identify atherosclerotic plaques with a high risk for sudden disruption before stroke or myocardial infarction would be of great utility. We used a rabbit model of controlled atherothrombosis to test whether in vivo MRI can noninvasively distinguish between plaques that disrupt after pharmacological triggering (vulnerable) and those that do not (stable). Methods and Results—Atherosclerosis was induced in male New Zealand White (n=17) rabbits by cholesterol diet and endothelial denudation of the abdominal aorta. After baseline (pretrigger) MRI with and without gadolinium contrast, the rabbits underwent 2 pharmacological triggerings to induce atherothrombosis, followed by another MRI 48 hours later (post-triggering). Atherosclerosis was identified by the pretriggered images in all rabbits, and thrombosis was identified in 9 of 17 animals (53%) by post-trigger MRI. After the animals were euthanized, 95 plaques were analyzed; 28 (29.5%) had thrombi (vulnerable) and 67 did not (stable) (70.5%). Pretriggered MRI revealed comparable stenosis in stable and vulnerable plaques, but vulnerable plaques had a larger plaque area (4.8±1.6 versus 3.0±1.0 mm2; P=0.01), vessel area (9.2±3.0 versus. 15.8±4.9 mm2; P=0.01), and higher remodeling ratio (1.16±0.2 versus 0.93±0.2; P=0.01) compared with stable plaques. Furthermore, vulnerable plaques more frequently exhibited (1) positive remodeling (67.8% versus 22.3%; P=0.01), in which the plaque is hidden within the vessel wall instead of occluding the lumen; and (2) enhanced gadolinium uptake (78.6% versus 20.9%; P=0.01) associated with histological findings of neovascularization, inflammation, and tissue necrosis. Conclusions—We demonstrate that in vivo MRI at 3.0 T detects features of vulnerable plaques in an animal model of controlled atherothrombosis. These findings suggest that MRI may be used as a noninvasive modality for localization of plaques that are prone to disruption.

Diffusion properties of NAA in human corpus callosum as studied with diffusion tensor spectroscopy.

In diffusion tensor imaging (DTI) the anisotropic movement of water is exploited to characterize microstructure. One confounding issue of DTI is the presence of intra‐ and extracellular components contributing to the measured diffusivity. This causes an ambiguity in determining the underlying cause of diffusion properties, particularly the fractional anisotropy (FA). In this study an intracellular constituent, N‐acetyl aspartate (NAA), was used to probe intracellular diffusion, while water molecules were used to probe the combined intra‐ and extracellular diffusion. NAA and water diffusion measurements were made in anterior and medial corpus callosum (CC) regions, which are referred to as R1 and R2, respectively. FA(NAA) was found to be greater than FA(Water) in both CC regions, thus indicating a higher degree of anisotropy within the intracellular space in comparison to the combined intra‐ and extracellular spaces. A decreasing trend in the FA of NAA and water was observed between R1 and R2, while the radial diffusivity (RD) for both molecules increased. The increase in RD(NAA) is particularly significant, thus explaining the more significant decrease in FA(NAA) between the two regions. It is suggested that diffusion tensor spectroscopy of NAA can potentially be used to further characterize microscopic anatomic organization in white matter. Magn Reson Med 58:1045–1053, 2007.

The Relationship of Ectopic Lipid Accumulation to Cardiac and Vascular Function in Obesity and Metabolic Syndrome

Storage of lipid in ectopic depots outside of abdominal visceral and subcutaneous stores, including within the pericardium and liver, has been associated with obesity, insulin resistance, and cardiovascular risk. We sought to determine whether anatomically distinct ectopic depots were physiologically correlated and site‐specific effects upon cardiovascular function could be identified. Obese subjects (n = 28) with metabolic syndrome but without known atherosclerotic disease and healthy controls (n = 18) underwent magnetic resonance imaging (MRI) and proton MR spectroscopy (MRS) to quantify pericardial and periaortic lipid volumes, cardiac function, aortic compliance, and intrahepatic lipid content. Fasting plasma lipoproteins, glucose, insulin, and free‐fatty acids were measured. Pericardial and intrahepatic (P < 0.01) and periaortic (P < 0.05) lipid volumes were increased in obese subjects vs. controls and were strongly and positively correlated (P ≤ 0.01) but independent of BMI (P = NS) among obese subjects. Intrahepatic lipid was associated with insulin resistance (P < 0.01) and triglycerides (P < 0.05), whereas pericardial and periaortic lipid were not (P = NS). Periaortic and pericardial lipid positively correlated to free‐fatty acids (P ≤ 0.01) and negatively correlated to high‐density lipoprotein (HDL) cholesterol (P < 0.05). Pericardial lipid negatively correlated to cardiac output (P = 0.03) and stroke volume (P = 0.01) but not to left ventricular ejection fraction (P = 0.46). None of the ectopic depots correlated to aortic compliance. In conclusion, ectopic storage of lipid in anatomically distinct depots appeared tightly correlated but independent of body size. Site‐specific functional abnormalities were observed for pericardial but not periaortic lipid. These findings underscore the utility of MRI to assess individual differences in ectopic lipid that are not predictable from BMI.

The effects of librations on the 13C chemical shift and 2H electric field gradient tensors in β-calcium formate

The magnitudes and orientations of the principal elements of the 13C chemical shift anisotropy (CSA) tensor in the molecular frame of the formate ion in β-calcium formate is determined using one-dimensional dipolar-shift spectroscopy. The magnitudes of the principal elements of the 13C CSA tensor are σ11C=104 ppm, σ22C=179 ppm, and σ33C=233 ppm. The least shielding element of the 13C CSA tensor, σ33C, is found to be collinear with the C–H bond. The temperature dependence of the 13C CSA and the 2H quadrupole coupling tensors in β-calcium formate are analyzed for a wide range of temperature (173–373 K). It was found that the span of the 13C CSA and the magnitude of the 2H quadrupole coupling interactions are averaged with the increasing temperature. The experimental results also show that the 2H quadrupole coupling tensor becomes more asymmetric with increasing temperature. A librational motion about the σ22C axis of the 13C CSA tensor is used to model the temperature dependence of the 13C CSA tensor. The t...