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Changes of membrane fluidity in erythrocytes of obese children: a spin label study.

Red blood cells depend on circulating lipoproteins for the turnover of membrane phospholipids. Thus abnormalities in lipid and lipoprotein metabolism are frequently associated with changes in composition and fluidity of erythrocyte membranes. Using the electron spin resonance technique we studied the physical state of erythrocyte membranes from 15 obese children with a body mass index ranging from 22.8 to 34 kg . m-2 and slight alterations of plasma lipids. The spin labels used, 5-doxylstearate and 16-doxylstearate, have shown an increase of the order parameter and of the correlation time, respectively. These observations indicate a decrease of fluidity at the surface and in the hydrophobic core of erythrocyte membranes from obese subjects. The results are statistically significant and they are discussed in relation to clinical aspects of childhood obesity. Studies on erythrocyte membrane molecular organization could represent a sensitive way to find out small metabolic alterations hardly detectable with compositional studies.

Adolescent↗

Conformational equilibria in spin-labeled hemoglobin.

A component characteristic of deoxyhemoglobin appears in the paramagnetic resonance spectrum of spin-labeled oxyhemoglobin, and vice versa, under conditions of pH and ionic strength consistent with the interpretation that the spectrum is sensitive to the conformational equilibrium of the carboxy-terminal histidines. The oxygenation-induced change in the resonance spectrum is discussed in terms of shifts in this equilibrium.

Chemical Phenomena↗

Morphological, contrast-enhanced and spin labeling perfusion imaging for monitoring of relapse after RF ablation of renal cell carcinomas.

MR perfusion imaging was applied for the assessment of completeness in the destruction of renal cell carcinomas by RF ablation (RFA) in a pilot study. An arterial spin labeling (ASL) approach was compared to conventional contrast-enhanced T1-weighted (CE-T1w) imaging. Ten patients suffering from renal cell carcinoma were treated by RFA. For the assessment of the extent of coagulation and for the detection of residual tumor, T1-weighted gradient-echo imaging, T2-weighted spin echo imaging and two different perfusion imaging techniques were performed before, 1 day and 6 weeks after RFA at 1.5 T. Perfusion imaging comprised CE-T1 weighted and FAIR-TrueFISP ASL imaging. Perfusion images recorded in the acute stage after RFA showed higher compliance to the definitive ablation volume reached after 6 weeks than T2-weighted images, which underestimated the true necrosis size. In the detection of residual tumor tissue, both modalities complimented each other. The exclusion of residual tumor tissue could more reliably be performed using perfusion-imaging methods. Both perfusion-imaging modalities showed sufficient imaging quality for post-interventional monitoring. Perfusion imaging provides a higher predictability of the completeness of tumor ablation and extent of coagulation than T2-weighted imaging alone. Since the results of the FAIR-TrueFISP sequence are promising, the administration of potentially nephrotoxic contrast media may be avoided in the respective patient cohort.

Aged↗

Bayesian inference of hemodynamic changes in functional arterial spin labeling data.

The study of brain function using MRI relies on acquisition techniques that are sensitive to different aspects of the hemodynamic response contiguous to areas of neuronal activity. For this purpose different contrasts such as arterial spin labeling (ASL) and blood oxygenation level dependent (BOLD) functional MRI techniques have been developed to investigate cerebral blood flow (CBF) and blood oxygenation, respectively. Analysis of such data typically proceeds by separate, linear modeling of the appropriate CBF or BOLD time courses. In this work an approach is developed that provides simultaneous inference on hemodynamic changes via a nonlinear physiological model of ASL data acquired at multiple echo times. Importantly, this includes a significant contribution by changes in the static magnetization, M, to the ASL signal. Inference is carried out in a Bayesian framework. This is able to extract, from dual-echo ASL data, probabilistic estimates of percentage changes of CBF, R(2) (*), and the static magnetization, M. This approach provides increased sensitivity in inferring CBF changes and reduced contamination in inferring BOLD changes when compared with general linear model approaches on single-echo ASL data. We also consider how the static magnetization, M, might be related to changes in CBV by assuming the same mechanism for water exchange as in vascular space occupancy.

Bayes Theorem↗

Membrane-mediated assembly of annexins studied by site-directed spin labeling.

Annexins are soluble proteins that bind to membranes in the presence of Ca2+. Crystal structures have been determined for some soluble forms, but little is known about the important membrane-bound state. We employed site-directed spin labeling to demonstrate that 1) annexin XII assumes a trimer configuration similar to the crystal structure when bound to bilayers under physiological conditions; 2) trimer assembly on bilayers is remarkably rapid, occurring on a millisecond time scale, whereas subunit exchange requires hours; and 3) different annexins can mix to form heterotrimers. The rapid assembly and heterotrimer formation have important implications concerning the cellular functions of annexins.

Annexins↗

Cerebral vascular response to hypercapnia: determination with perfusion MRI at 1.5 and 3.0 Tesla using a pulsed arterial spin labeling technique.

PURPOSE: To compare the quantification of cerebral blood flow (CBF) at 1.5 and 3.0 Tesla, under normo- and hypercapnia, and to determine the cerebral vascular response (CVR) of gray matter (GM) to hypercapnia, a pulsed arterial spin labeling technique was used. Additionally, to improve GM CBF quantification a high-resolution GM-mask was applied. MATERIALS AND METHODS: CBF was determined with the QUIPSS II with thin slice TI1 periodic saturation (Q2TIPS) sequence at 1.5 and 3.0 Tesla in the same group of eight subjects, both under normocapnia and hypercapnia. Absolute GM-CBF maps were calculated using a GM-mask obtained from a high-resolution structural scan by segmentation. The CVR to hypercapnia was derived from the quantitative GM-CBF maps. RESULTS: For both field strengths, the GM-CBF was significantly higher under hypercapnia compared to normocapnia. For both conditions, there was no significant difference of GM-CBF for 1.5 and 3.0 Tesla; the same applies to the CVR, which was 4.3 and 4.5%/mmHg at 1.5 and 3.0 Tesla, respectively. CONCLUSION: The method presented allows for the quantification of CBF and CVR in GM at the common clinical field strengths of 1.5 and 3.0 Tesla and could therefore be a useful tool to study these parameters under physiological and pathophysiological conditions.

Adult↗

Internalization of the spin-labelled surface potential probe CAT12 by energized mitochondria.

Rat liver mitochondria briefly incubated in the presence of 50 microM 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12) bind 1.9 nmol of this spin-labelled membrane probe per mg mitochondrial protein in a form which is not reducible by ascorbate. Upon energization with ATP the amount of the non-reducible CAT12 increases by about 37%. It is concluded that CAT12 which is not reducible by externally added ascorbate is bound to the inner surface of the inner mitochondrial membrane and/or accumulated in the matrix compartment. Therefore, CAT12 is not suitable to monitor the surface potential of mitochondria and other organelles which develop a transmembrane potential, negative inside.

Adenosine Triphosphate↗

Efficient visualization of vascular territories in the human brain by cycled arterial spin labeling MRI.

Intracranial vascular territories are usually visualized with the use of angiographic techniques. However, because it is difficult to visualize the distal vascular bed with any angiographic technique, it is also difficult to infer the parenchymal borders of vascular territories. Arterial spin labeling (ASL) MRI provides information on cerebral perfusion, and a regional ASL (regASL) approach offers the potential to visualize perfusion of the anterior and posterior circulation separately. Current techniques perform the labeling in each feeding artery as a separate experiment, which is very time-consuming. In this work a very time-efficient regASL technique is presented that acquires all vascular territories within the same experiment. Images with different combinations of labeled arteries are combined to delineate the vascular territory of interest. Five subjects were examined with a clinical 1.5T MR scanner. A sharp delineation of the middle cerebral artery (MCA) and posterior cerebral artery (PCA) territories with whole-brain coverage was achieved in all subjects. The resulting signal-to-noise ratios (SNRs) for conventional regASL and the proposed cycled regASL were 9.3+/-2.4 and 15.3+/-5.1, respectively. An optimized setup was achieved by combining the regional labeling scheme with an efficient readout technique, which yielded a total measurement time of 2 min for three vascular territories.

Algorithms↗

Imaging blood flow in brain tumors using arterial spin labeling.

Measurements of tumor blood flow (TBF) are important for understanding tumor physiology and can be valuable in selecting and evaluating therapies. Brain tumors typically present reduced blood flows compared to normal brain tissue. This study shows that the arterial spin labeling (ASL) technique can be used to measure TBF non-invasively in a rat glioma model. Results show that TBF in the core (36.3 +/- 18.9 ml/100g/min, n=4) and peripheral regions (85.3 +/- 26.9 ml/100g/min, n=4) of the tumor are significantly reduced and show considerable heterogeneity compared to cerebral blood flow (CBF) of normal brain tissue (147.7 +/- 31.1 ml/100g/min, n=4), while T(1) in the tumor (2.6 +/- 0.1 sec) is significantly elevated compared to normal tissue T(1) (2.0 +/- 0.0 sec). These results strongly support the feasibility of using the ASL technique to evaluate different cancer treatment strategies, to monitor the effects of agents designed to modulate TBF and oxygenation (e.g., carbogen gas), and to assess and guide the use of anti-angiogenic agents. Magn Reson Med 44:169-173, 2000.

Animals↗

Spin-label assay for phospholipase A2.

A rapid and continuous method for measuring phospholipase A2 activity using electron spin resonance spectroscopy and a spin-labeled phospholipid as a substrate has been developed. The substrate, 1-palmitoyl-2-(4-doxylpentanoyl)glycerophosphocholine, gives rise principally to a broad ESR line in aqueous solution due to strong spin-spin interactions, probably resulting from its micellar formation. Upon addition of bee venom phospholipase A2, the water-soluble product, 4-doxylpentanoic acid, is released which brings about a sharp three-line spectrum. Thus, the kinetics of phospholipase A2 activity can be followed by monitoring the increase in the ESR signal amplitude of the three-line spectrum, which is linearly proportional to the amount of 4-doxylpentanoic acid produced; no separation of the product from the substrate is needed during the measurement. The rate of hydrolysis of 1 nmol min-1 can be accurately measured within a 5-min period of time in a sample volume of 100 microliters. This new method should be useful for assaying phospholipase A2 activities in various biological systems.

Bee Venoms↗

A transmembrane form of annexin XII detected by site-directed spin labeling.

Previous studies of the annexin family of Ca2+ binding proteins identified a soluble monomer in the absence of Ca2+ and a trimer adsorbed on the membrane surface in the presence of Ca2+. On the basis of site-directed spin-labeling studies of annexin XII at low pH, we now report a membrane-inserted form of the protein with a dramatically different structure. The data suggest that upon insertion a continuous transmembrane alpha-helix is reversibly formed from a helix-loop-helix motif in the solution structure. Other regions with similar membrane-insertion potential were identified in the amino acid sequence, and we propose that the corresponding helices come together to form an aqueous pore that mediates the ion channel activity reported for several annexins.

Amino Acid Sequence↗

Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone.

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the alpha-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg(2+) shifts it in the opposite direction. Relaxation enhancement, due to Ni(2+) ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.

Amino Acid Sequence↗

Structural features of the C-terminal domain of bovine rhodopsin: a site-directed spin-labeling study.

Eleven single-cysteine substitution mutants have been prepared in the sequence 325-340 of rhodopsin, corresponding to the C-terminal domain. Each of the cysteine mutants was modified with a selective nitroxide reagent to introduce a spin-labeled side chain. The electron paramagnetic resonance spectra of the labeled proteins were analyzed in terms of side chain dynamics. At all sites, the spectra reflected the presence of two populations of different mobility, although one was always dominant. The mobility of the dominant population increased in a regular fashion from the palmitoylation sites at 322C and 323C to the C-terminus, where the spectra resembled those of an unfolded protein. This apparent mobility gradient is only slightly affected in mutants lacking the palmitoyl groups, suggesting that they are not responsible for physically anchoring the C-terminal peptide at one end. Binding of a monoclonal antibody to its epitope at the C-terminus dramatically reduces the mobility of nearby residues, creating a local mobility gradient opposite that in the absence of the antibody. These results indicate that the C-terminal domain of rhodopsin, beyond the palmitoylation sites, is highly disordered and dynamic, resembling an unfolded peptide tethered at one end.

Amino Acid Sequence↗

Continuous arterial spin labeling perfusion measurements using single shot 3D GRASE at 3 T.

Single shot 3D GRASE is less sensitive to field inhomogeneity and susceptibility effects than gradient echo based fast imaging sequences while preserving the acquisition speed. In this study, a continuous arterial spin labeling (CASL) pulse was added prior to the single shot 3D GRASE readout and quantitative perfusion measurements were carried out at 3 T, at rest and during functional activation. The sequence performance was evaluated by comparison with a CASL sequence with EPI readout. It is shown that perfusion measurements using CASL GRASE can be performed safely on humans at 3 T without exceeding the current RF power deposition limits. The maps of resting cerebral blood flow generated from the GRASE images are comparable to those obtained with the 2D EPI readout, albeit with better coverage in the orbitofrontal cortex. The sequence proved effective for functional imaging, yielding time series of images with improved temporal SNR with respect to EPI and group activation maps with increased significance levels. The method was further improved using parallel imaging techniques to provide increased spatial resolution and better separation of the gray-white matter cerebral blood flow maps.

Adult↗

A spin label study of the perturbation effect of tertiary amine anesthetics on brain lipid liposomes and synaptosomes.

The membrane disordering efficiency of four local anesthetics, including lidocaine, tetracaine, dibucaine and heptacaine (piperidinoethyl ester of 2-heptyloxyphenylcarbamic acid) has been studied by spin-labeling methods. The disordering efficiency of the drugs in rat total brain lipid liposomes was quantitated with the initial slope value of the order parameter versus drug concentration curve, the so-called change-in-order parameter value. Using the positional isomers of m-doxyl stearic acids (m = 5, 12 and 16), it has been demonstrated that the tested drugs reveal quite different disordering efficiency. There is a clear tendency of increasing disordering efficiency towards the methyl terminal of the lipid acyl chains. By a comparison of order parameter versus drug concentration and temperature at three depths of rat brain total lipid liposomes and synaptosomes, it is shown that the 'fluidizing effect' of local anesthetics does not correspond to fluidization of membrane by temperature and that tetracaine and dibucaine do not have equal disordering efficiency as judged by their solubility in the membrane. The disordering efficiency of these drugs on the hydrocarbone core of a membrane qualitatively corresponds to their anesthetic potency. Similar results were obtained in liposomes and synaptosomes. It is assumed that there is a similar incorporation of the local anesthetics in the liposomes and in the lipid part of synaptosomes.

Anesthetics, Local↗

Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence, spin label and SAXS study. Single angle X-ray scattering.

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.

Anesthetics, Local↗

Spin labeling studies of membrane proteins in erythrocyte ghosts from patients with Huntington's disease.

Previous electron spin resonance (ESR) studies employing a protein-specific spin label have suggested an alteration in membrane proteins in erythrocytes in Huntington's disease (HD). Recently, Beverstock and Pearson [4] have published the results of an ESR study not confirming our findings. In the present communication, we show that with correct use of the statistical technique of the Dixon test to determine if extreme values can be eliminated, one of the data points of these authors can be rejected. Recalculation of their results suggests a significant difference in the physical state of membrane proteins in HD erythrocytes, confirming our findings.

Electron Spin Resonance Spectroscopy↗

[Effect of temperature and binding with copper ions on the motility of spin-labeled oxy- and methemoglobin subunits].

Using the method for separate determination of correlation times of spin-labeled proteins (tau M) and labels (tau R) it has been shown that at temperatures below and about 25 degrees the mobility of oxy Hb subunits is higher than that of met Hb. From 30 degrees on oxy and met Hb show identical flexibility (tau M = 40 ns) in 0.01 M phosphate buffer (pH 7.3) containing 0.15 M NaCl. With a decline in pH from 7.3 to 6.4 the intramolecular mobility of met Hb subunits decreases. In the absence of 0.15 M NaCl at pH 7.3 (5 degrees C) met Hb becomes more flexible (tau M drops from 25 to 16 ns). Complex formation of beta-chains of oxy Hb with one Cu+2 ion at 20 degrees has a negligible bearing on the flexibility of the protein, whereas addition of second ion considerably enhances interaction between the subunits of the tetramer and decreases its flexibility (tau M rises from 17 to 30 ns).

Copper↗