Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medicationfree adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. 1H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3 ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3 ppm peak intensity and a discontinuity at ~18 °C, for which the simplest explanation is a phase transition from Ld to Lo phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3 ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance

Ultraviolet radiation reduces desmosine cross-links in elastin

Elastic fibers, a major component of the extracellular matrix of the skin, are often exposed to ultraviolet (UV) radiation throughout mammalian life. We report on an in vitro study of the alterations in bovine nuchal ligament elastic fibers resulting from continuous UV-A exposure by the use of transmission electron microscopy (TEM), histology, mass spectrometry, and solid state 13C NMR methodologies. TEM images reveal distinct cracks in elastic fibers as a result of UV-A irradiation and histological measurements show a disruption in the regular array of elastic fibers present in unirradiated samples; elastic fibers appear shorter, highly fragmented, and thinner after UV-A treatment. Magic angle spinning 13C NMR was applied to investigate possible secondary structural changes or dynamics in the irradiated samples; our spectra reveal no differences between UV-A irradiated and non-irradiated samples. Lastly, MALDI mass spectrometry indicates that the concentration of desmosine, which forms cross-links in elastin, is observed to decrease by 11 % following 9 days of continuous UV-A irradiation, in comparison to unirradiated samples. These alterations presumably play a significant role in the loss of elasticity observed in UV exposed skin

Structure and dynamics studies by solid-state NMR spectroscopy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2002.Vita.Includes bibliographical references.The major goal of this work is the development of high resolution solid state 205T1 NMR techniques and their application to the elucidation of the mechanism and dynamics of ion exchange in biological solids. The thesis starts with a review of recent progress in solid state NMR spectroscopy. The importance of directly observing ions in biological systems is discussed, and the advantages of 205T1 spectroscopy are noted of. Theoretical framework of the NMR experiments is described in chapter 2. The third chapter presents the development of methodology for solid state 205T1 NMR. The difficulties of 205T1 NMR are identified, and ways to circumvent the limitations are discussed. Several model compounds convenient for calibration of the key experimental parameters are discussed. The 205T1 direct excitation and H-205T1 cross polarization experiments are demonstrated. The fourth chapter describes the application of 205T1 high resolution solid state NMR to the investigation of the behavior of a small ion channel in the liquid crystalline phase. Here, thallium binding to gramicidin A in DMPC bilayers has been studied. Additional applications of 205T1 NMR to study liquid crystalline phases are also discussed. A summary and the future directions of the 205T1 NMR spectroscopy are presented in chapter 5.(cont.) The sixth chapter describes a 129Xe-19F high resolution solid state NMR study of crystalline XeF2. The absence of trapped Xe gas in crystalline XeF2 has been demonstrated. The 129Xe chemical shift anisotropy parameters have been extracted, and 19F-129Xe J-coupling has been measured. Progress in NMR spectroscopy is largely dependent on the capabilities and limitations of the experimental hardware. Chapter 7 presents a brief review of the development of probes for solid state NMR experiments. A triple channel H-13C-15N transmission line probe designed and constructed by the author is described.by Boris Arkadievich Itin.Ph.D

Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques

While solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for studying viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multiprotein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses. <i>In vitro</i> capsid assembly by combining individually purified, labeled, and unlabeled components in NMR quantities is not a viable option for most viruses. To overcome this barrier, we present a method that utilizes sequential protein expression and in cell assembly of component-specifically labeled viral capsids in amounts suitable for NMR studies. We apply this approach to purify capsids of bacteriophage ϕ6 isotopically labeled on only one of its four constituent protein components, the NTPase P4. Using P4-labeled ϕ6 capsids and the sensitivity enhancement provided by dynamic nuclear polarization, we illustrate the utility of this method to enable ssNMR studies of complex viruses

Elucidating the chemical structure of pyrogenic organic matter by combining magnetic resonance, mid-infrared spectroscopy and mass spectrometry

Fire-derived organic matter (pyrogenic organic matter, or PyOM), despite its apparent long term stability in the environment, has recently been reported to degrade faster than previously thought. Current studies have suggested that the composition and structure of PyOM can provide new insights on the mechanisms by which C and N from pyrolyzed biomaterials are stabilized in soils. To better understand the chemical structure of PyOM produced under typical fire conditions in temperate forests, samples of dual-enriched (13C/15N) Pinus ponderosa wood and the charred material produced at 450 °C were analyzed by solid state nuclear magnetic resonance (ssNMR), electron paramagnetic resonance (EPR), diffuse reflectance Fourier transform infrared (DRIFT) spectroscopy, and both isotopic and elemental composition (C, H, O, and N). Notably, the use of high magnetic field strengths in combination with isotopic enrichment augmented the NMR detection sensitivity, and thus improved the quality of molecular information as compared with previously reported studies of pyrogenic materials. The key molecular groups of pine wood and the corresponding PyOM materials were determined using magic-angle spinning (MAS) 13C, 15N, and 1H NMR. Together with DRIFT and EPR measurements, ssNMR revealed the formation of a free radical-containing disordered blend of nitrogenous aromatics and heat resistant aliphatics in the PyOM due to incomplete combustion of the precursor wood. 13C ssNMR and DRIFT analyses showed the removal of oxygenated aliphatics due to pyrolysis of the precursor wood and the dominant contribution of multiply-bonded and oxygenated aromatic structures in the resulting PyOM. However, the 18O isotopic analyses indicated selective retention of ligneous moieties during charring at 450 °C. 15N ssNMR studies implied that the nitrogenous species in PyOM corresponded to thermally altered rather than heat resistant domains of the pine wood precursor. Our molecular characterization suggests that biomaterials pyrolyzed near 450 °C may degrade in soils faster than those pyrolyzed at higher temperatures and may not represent a stable C sink in terrigenous ecosystems

Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. 1H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3 ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3 ppm peak intensity and a discontinuity at ~18 °C, for which the simplest explanation is a phase transition from Ld to Lo phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3 ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance

Sustainable fabrication of plant cuticle-like packaging films from tomato pomace agro-waste, beeswax, and alginate

Plant cuticles have been used as models to produce hydrophobic films composed of sodium alginate, the fatty acid fraction of tomato pomace agrowaste, and beeswax. The fabrication process consisted of the blending of components in green solvents (water and ethanol) and a subsequent thermal treatment (150 °C, 8 h) to polymerize unsaturated and polyhydroxylated fatty acids from tomato pomace. When sodium alginate and tomato pomace fatty acids were blended, free-standing films were obtained. These films were characterized to evaluate their morphological (SEM), chemical (solid-state NMR, ATR-FTIR), mechanical (tensile tests), thermal (TGA), and hydrodynamic (water contact angle, uptake, and permeability) properties. A comparison between nonpolymerized and polymerized samples was carried out, revealing that the thermal treatment represents a sustainable route to create structured, composite networks of both components. Finally, beeswax was added to the blend with the same amounts of sodium alginate and tomato pomace fatty acids. The presence of the wax improved the hydrophobicity and the mechanical and water barrier properties as well as decreased the water uptake. These results indicate that polymerized plant cuticle-like films have valuable potential for packaging applications