National Biomedical Electron Paramagnetic Resonance Center

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Research Highlight #122
Superoxide Generation from eNOS—The Role of Pterins

Jeannette Vasquez-Vivar
Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin

According to the American Heart Association's Heart Disease and Stroke Statistics – 2004 Update released in January, cardiovascular diseases (CVD) remain the #1 killer in the US. The report also shows that CVD is the No. 3 cause of death for children under age 15, behind certain conditions originating in the perinatal period and accidents. Cardiovascular diseases include high blood pressure, coronary heart disease (heart attack and angina), congestive heart failure, stroke, and congenital heart defects. We are examining the role of free radicals, nitric oxide and superoxide in the manifestation of endothelial dysfunction. This is considered an early clinical sign and risk factor associated with the progression of CVD. We have showed that the formation of nitric oxide and superoxide from endothelial nitric oxide synthase (eNOS), an enzyme expressed by the cells covering blood vessels, is tightly regulated by tetrahydrobiopterin. The clinical significance of this finding is indicated by the fact that supplementation with the cofactor ameliorates endothelial dysfunction in genetic models of CVD. Thus, the central research hypothesis is that diminished levels of tetrahydrobiopterin tip the balance in favor of eNOS-dependent superoxide generation setting up endothelial dysfunction. The causes or factors involved in the lowering of tetrahydrobiopterin in diseased blood vessels are not known. In order to examine this occurrence, we are developing new tools that allow us to understand the control of tetrahydrobiopterin balance at the molecular level. During the last year our efforts have been devoted to isolation of one of the proteins involved in the regulation of tetrahydrobiopterin synthesis. The GTP cyclohydrolase I feedback protein regulates the tuning of the tetrahydrobiopterin level in various cells. To examine its role in the regulation of tetrahydrobiopterin in blood vessels, the human gene encoding the protein was cloned and expressed in a bacterial system that permits the isolation of large amounts of recombinant protein. This preparation is being used to generate specific antibodies against the protein that can be used to detect the protein in cells and tissues. The first step has been successfully accomplished and we are now in the process of obtaining the antibodies. With these antibodies we will be able to compare the protein levels in healthy and diseased blood vessels. This information is of vital importance since we showed that in animal models with induced high cholesterol levels, tetrahydrobiopterin levels are low. The reason why high cholesterol decreases tetrahydrobiopterin levels however remains uncertain. We tested the possibility that oxidative stress, a condition associated with high cholesterol level, increases the expression of the inhibitory protein and in that way decreases tetrahydrobiopterin. Initial results indicate that oxidative stress in fact augmented expression of the inhibitory protein in endothelial cells. Our results also show that the inhibitory protein is augmented in response to particular triggers since it does not respond to inflammatory mediators, which are also considered to contribute to the progression of CVD. Our next step is to examine the correlation between levels of the inhibitory protein and the production of superoxide in the endothelium.

Research Highlight #121
HFSS Modeling of Aqueous Samples in Microwave Resonators for EPR

James S. Hyde, Richard R. Mett, and Jason W. Sidabras
Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin

Water has a very high dielectric constant and, in addition, very high dielectric loss. Placement of the EPR sample cuvette in the microwave resonator is extremely critical. The recent advent of improved finite-element software for modeling of electromagnetic field distributions — so-called High Frequency Structure Simulator (HFSS) — combined with the availability of much faster computers permits optimization of the design of an aqueous sample cuvette, which is hardly practical when repeated modifications and measurements are the only options. We report here the first examples of the use of HFSS in a context of aqueous samples for EPR.

There are two general principles for placement of aqueous cells in a microwave resonator: 1) localization near a node in the standing wave pattern where the RF electric field is zero, and 2) orientation of the sample cuvette such that the electric field is perpendicular to the surface, resulting in a discontinuity of the electric field vector and therefore in reduced dielectric loss. Using HFSS as well as analytical methods, we wrote in 2003 the first theoretical paper on aqueous samples in microwave cavities in the so-called "perpendicular" orientation (Mett et. al JMR 165:137-152, 2003). During the course of that work, a new aqueous sample cell geometry was discovered that holds the promise of increase in sensitivity by a factor of 5 to 6, which we feel is very exciting. Figure 1 illustrates the geometry for the rectangular TE102 cavity in the Y-Z plane (corresponding to indices 0, i.e. uniform field in the Y direction, and 2, i.e. 2 half wavelengths variation in the Z direction). The intensity and orientation of the RF electric field are indicated by arrows. The new geometry consists of parallel planar dielectric septa oriented perpendicular to the electric field with aqueous sample lying between septa. The number of such septa is large — 20 or more. A number of ways can be envisaged to construct such a cuvette, and this work is underway.

Figure 2 shows a loop gap resonator (LGR) geometry, and Fig. 3 zooms in on the sample loop. Water is contained in a star-shaped cuvette (we call it "AquaStar") with 8 vanes radiating from the center to the walls. The geometry has been specifically optimized using HFSS for X-band (9.5 GHz) for 10 microliters of sample. In Fig. 3, arrows indicate the direction of the electric field and shading the magnitude — dark is low, light is high. Electric field vectors are oriented perpendicular to the surfaces of all 8 vanes near the center of the sample loop. At the edge, 4 vanes lie in RF electric field nodes and 4 remain in perpendicular orientation. Thus, both principles for aqueous sample-cell design are active. We have determined that manufacture of the AquaStar cuvette is feasible using multilumen extruded plastic techniques. The targeted application for the geometry of Figs. 2, 3 is site specific spin labeling, SDSL, for molecular structure determination.



Research Highlight #120
Formation of Cholesterol-Glycosphingomyelin Raft-Domains in Unsaturated Phosphatidylcholine membranes and Their Characterization

Witold K. Subczynski1, Aki Kusumi2, and James S. Hyde1
1Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin; 2Department of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya, Japan

The cell membrane has a 2-dimensional liquid-like structure, but it is not a simple liquid. Rather, it is a non-ideal liquid mixture of various molecules, which contains domain structures of various time scale and space scale that are forming and dispersing continuously within the cell membrane. Rafts are this type of membrane domains that require lipid interactions for their formation. The interest in rafts has surged in the majority of biomedical fields recently because they may play an important role in signal transduction, protein sorting, and pathogen action. We would like to understand the molecular organization and dynamics in these raft-domains and to provide new insight into the formation mechanism. We developed the method called the discrimination by oxygen transport (DOT) method which uses the pulse saturation recovery electron paramagnetic resonance (SR EPR) spectroscopy. Using this method it is possible not only to indicate the presence of raft domains (which is not an easy task) in model and biological membranes, but also characterize their physical properties, namely oxygen transport at different depths in the membrane. These measurements can be done simultaneously for the raft and the surrounding bulk membrane without need for separation. Using this method we discriminated and characterized the raft domain in the influenza virus envelope membrane as a very tightly packed structure with slow oxygen transport but rather high lipid exchange rate with the surrounding bulk lipids. Presently we are focusing our research on model membranes made of raft forming lipid mixtures to better understand how lipid composition and surrounding physical conditions affect the size, lifetime and dynamics of the raft-constituent molecules and the raft itself in the membrane. We believe that our work will help to better understand how membrane dynamic structure regulate cell membrane fundamental functions, and, would eventually contribute to better medical treatments in the future.


Research Highlight #119
Identification of a Membrane-Associated a-Helix in Myelin Basic Protein

Ian Bates1, Jimmy B. Feix2, Joan Boggs1, and George Harauz1
1Department of Molecular Biology and Genetics and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada; 2Department of Biophysics and National Biomedical EPR Center, Medical College of Wisconsin, Milwaukee, Wisconsin

Multiple sclerosis (MS) is thought to be an autoimmune disease characterized by chronic inflammatory response against myelin in the central nervous system (CNS). There is significant evidence that myelin basic protein (MBP) is a candidate antigen for T-cells and autoantibodies in MS. The level of anti-MBP antibodies is increased in the cerebrospinal fluid (CSF) of patients with active MS, as well as in 96% of patients with relapsing and chronic MS. MBP maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers. The mechanism and sites that are important for membrane adhesion are not known.

A region of between Proline 85 and Proline 96 (human sequence numbering) was identified to be a minimal epitope for T-lymphocytes that recognize MBP. Experimental treatments for MS based on peptide mimetics of MBP have focused on this region of the protein. We have used molecular dynamics and site-directed spin labeling (SDSL) EPR to demonstrate that this antigenic segment, when bound to a myelin-like membrane surface, forms an amphipathic a-helix with side chains along one surface penetrating up to 12 Å into the bilayer. The helix is tilted ~9o with respect to the membrane surface, and a central lysine residue is in an ideal position for electrostatic interaction with the negatively charged phospholipid head groups.

1. Bates, I. R., Boggs, J. M., Feix, J. B., and Harauz, G. "Membrane anchoring and charge effects in the interaction of myelin basic protein (MBP) with lipid bilayers studied by site-directed spin labeling," J. Biol. Chem. 278, 29041-29047 (2003).
2. Bates, I. R., Feix, J. B., Boggs, J. M., and Harauz, G. "An immunodominant epitope of myelin basic protein is an amphipathic a-helix." J. Biol. Chem. 279, 5757-5764 (2004).
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