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PubMed1987

Preparation of spin-labelled sulfatides for EPR studies on model membranes.

Using the N-hydroxysuccinimide ester of the fatty acids, galactosylceramide I3-sulfate containing a 5-or 16-doxyl-stearoyl residue was prepared in good yield by acylation of the galactosylsphingosine I3-sulfate (lysosulfatide) obtained from the saponification of the bovine brain sulfatide. The EPR behavior of the two semisynthetic sulfatides was analyzed in natural sulfatide micelles and in multilamellar vesicles of egg phosphatidylcholine. The evaluated parameters demonstrate that these spin-labelled sulfatides can be used for the study of sulfatide behavior in lipid structures.

Animals↗
PubMed2007

Electron paramagnetic resonance spectroscopy of site-directed spin labels reveals the structural heterogeneity in the N-terminal domain of apoA-I in solution.

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Apolipoprotein A-I↗
PubMed1997

Estimation of water extraction fractions in rat brain using magnetic resonance measurement of perfusion with arterial spin labeling.

The model used for calculating perfusion by MRI techniques that use endogenous water as a tracer assumes that arterial water is a freely diffusible tracer. Evidence shows that this assumption is not valid in the brain at high blood flow rates, at which movement of water into and out of the microvasculature becomes limited by diffusion across the blood-brain barrier. In this work, the arterial spin-labeling technique is used to show that fraction of arterial water that is dependent on blood flow rate remains in the vasculature and does not exchange with brain tissue water. By using perfusion measurements without and with magnetization transfer (MT) effects, one can distinguish arterial label that exchanges into tissue because blood has much smaller MT than brain tissue. Using this technique, the extraction fraction for water is measured in the rat brain at various cerebral blood flow rates. At high flow rates (approximately 5 ml/g/min), the extraction fraction for water is found to be about 45% in rat brain. Disruption of the blood-brain barrier with D-mannitol caused an increase in the extraction fraction for water. It was possible to form an image related to the extraction fraction for water. The ability to estimate the amount of vascular water exchanging with tissue water by MRI may represent a noninvasive approach to detect the integrity of the blood-brain barrier.

Animals↗
PubMed1982

The role of possible membrane damage in porphyria cutanea tarda: a spin label study of rat liver cell membranes.

Two groups of rats were made porphyric by treatment for a shorter or a longer time with hexachlorobenzene (HCB); a third group was subjected to chronic treatment with ethanol; the fourth group comprised untreated, control animals. Suspensions were prepared of hepatocytes isolated from the livers of the animals in the individual groups, and in these systems the cell membranes were studied by spin labeling. In all three chronically treated groups, the order parameter calculated from the ESR spectra was significantly lower than that of the control group. The order parameters ofr the first three treated groups, however, did not differ significantly from one another. If either HCB or ethanol was incorporated into the isolated hepatocytes of the healthy control animals, the fluidity of the hepatocyte membranes increased. The order parameter decrease (compared to the control) for the hepatocyte membranes of the animals treated chronically with HCB or ethanol can be ascribed to the direct membrane-fluidizing effect of HCB or ethanol, and also to the altered lipid metabolism. With regard to the difference in the mechanisms of action of HCB and ethanol, on the basis of the experimentally proved membrane-damaging effect of the porphyrinogenic HCB it is probable that, besides other factors, the membrane damage may play an important role in the pathogenesis of porphyria cutanea tarda.

Animals↗
PubMed1998

Multisection cerebral blood flow MR imaging with continuous arterial spin labeling.

A method was evaluated to control for off-resonance saturation in noninvasive magnetic resonance imaging with continuous arterial spin labeling of cerebral blood flow. In phantoms and humans, application of amplitude-modulated radio-frequency irradiation during the control image corrected for saturation across the whole brain and made possible cerebral blood flow imaging in multiple sections at arbitrary angles to the labeling plane.

Adult↗
PubMed2004

Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding. A site-directed spin labeling study.

The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.

Amino Acid Sequence↗
PubMed1981

Interaction of superoxide dismutase with phospholipid liposomes. An uptake, spin label and calorimetric study.

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu2+, Zn2+ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7 degrees C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.

Animals↗
PubMed1988

Evidence for multiple conformational changes in the active center of thrombin induced by complex formation with thrombomodulin: an analysis employing nitroxide spin-labels.

Thrombomodulin (TM) is an endothelial cell surface protein that binds thrombin to form a reversible complex with altered enzyme specificity. The complex rapidly converts protein C to the anticoagulant enzyme activated protein C and has decreased fibrinogen clotting activity. To investigate whether formation of this complex elicits conformational changes in the active center of thrombin, we employed the following fluorosulfonyl spin-label inhibitors: N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-m-(fluorosulfonyl)benzamide (m-V); O-(2,2,6,6-tetramethyl-1-oxy-4-piperidinyl) N-[m-(fluorosulfonyl)phenyl]carbamate (m-VI); N-[4-(fluorosulfonyl)phenyl]-2,2,5,5-tetramethyl-1-oxy-3-pyrroline -3-carboxamide (p-I); N-(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-p-(fluorosulfonyl)benzamide (p-V). To compare the spectra of the free thrombin with those of the complex, the viscosity of the solution was adjusted with sucrose to give similar tumbling rates (isokylindric spectra) or the macromolecular rotational contribution to the spectra was essentially eliminated with saturated sucrose. Both a buffer-soluble proteolytic derivative of TM and the intact molecule elicited changes in the electron spin resonance signals of many of the labeled thrombins employed. Two of the labels, p-I and p-V, had previously been shown to exhibit decreased mobility when indole derivatives were bound to thrombin. When TM complexes with thrombin, the mobility of the p-I label increases while the mobility of the p-V label decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Synthesis and study of a spin-labeled cyclophosphamide analogue, 3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide.

3-(1-Oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (7) was isolated in 36% yield following H2O2-Na2WO4 oxidation of 3-(2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (6), which was synthesized in three steps (25% yield) starting with 4-amino-2,2,6,6-tetramethylpiperidine. Binding of 7 to mouse liver microsomes was investigated by optical and electron spin resonance spectroscopy. Compared with the mouse liver microsomal metabolism of 1, separate incubations of 6 and an ca. 1:1 mixture of 1 and 6 gave approximately 90 and 60% less acrolein, respectively. A spin-labeled metabolite of 7, viz., N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)phosphoramide mustard (9), was synthesized and its intramolecular O-alkylation at pH 7.4, 37 degrees C, was studied by 31P NMR spectroscopy. Compounds 7 and 9 were inactive in screening tests against L1210 lymphoid leukemia in mice.

Acrolein↗
PubMed2001

Nitric oxide and free stable nitroxyl radicals in the mechanism of biological action of the spin-labeled compounds.

A comparison of more important physical, chemical and biological properties of the nitric oxide (NO) and free stable nitroxyl radicals (nitroxides) on the base of their structural similarity is made in the article. The active moiety in the nitroxide molecule represents a sterically hindered nitric oxide. The mechanisms of biological action of the nitroxides and especially of their derivatives with antitumor agents from the groups of nitrogen mustards, nitrosoureas, aziridines and triazenes (spin-labeled compounds) is explained through the biological activities of sterically hindered NO. Similarly to NO, nitroxides also can react with superoxide anion radical (O(2)(-)), they possess superoxide dismutase (SOD) mimetic action. While the interaction of NO with O(2)(-)yields very toxic peroxynitrite (ONOO(-)), its formation is strongly limited in the presence of a nitroxide. It is known that the nitrosourea antitumor drugs, like lomustine (CCNU) and carmustine (BCNU), showed high general toxicity, one of the reasons for that probability is the formation of NO, and subsequently of ONOO(-), during their metabolism. The biological investigations of the nitroxides showed their considerably lower general toxicity that could be explained with the SOD-mimetic action of the nitroxide present in their molecule.

Antineoplastic Agents↗
PubMed1987

Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosinetriphosphatase in membranes from Squalus acanthias.

The sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)-ATPase] in membranous preparations from Squalus acanthias has been spin-labeled on sulfhydryl groups after prelabeling with N-ethylmaleimide. Saturation-transfer electron spin resonance spectroscopy has been used to study the rotational motions of the labeled protein on the microsecond time scale. Effective rotational correlation times deduced from the diagnostic line-height ratios in the second-harmonic, 90 degrees out-of-phase (V2') spectra are much larger than those deduced from the spectral integrals, indicating the presence of large-scale segmental motions, in addition to rotation of the protein as a whole. Experiments involving controlled cross-linking of the protein by glutaraldehyde, as well as measurements of the line broadening of the conventional electron spin resonance spectra, support this interpretation. Both the spectral integrals and diagnostic line-height ratios are found to increase irreversibly with time on incubation at temperatures greater than 20 degrees C, corresponding to a decrease in the segmental motion of the protein and probably also in the overall protein rotation. The native enzyme displays a marked nonlinearity in the Arrhenius temperature dependence of the activity at temperatures above 20 degrees C, and the activity decreases with a half-life of ca. 70 min on incubation at 37 degrees C (but not on incubation at low temperature), paralleling the time- and temperature-dependent changes in the saturation-transfer spectra of the labeled protein. Both of these observations suggest that the changes observed in the molecular dynamics could correspond to functional properties of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Arterial spin labeling perfusion fMRI with very low task frequency.

Functional magnetic resonance imaging (fMRI) has become the most widely used modality for visualizing regional brain activation in response to sensorimotor or cognitive tasks. While the majority of fMRI studies have used blood oxygenation level-dependent (BOLD) contrast as a marker for neural activation, baseline drift effects result in poor sensitivity for detecting slow variations in neural activity. By contrast, drift effects are minimized in arterial spin labeling (ASL) perfusion contrast, primarily as a result of successive pairwise subtraction between images acquired with and without labeling. Recent data suggest that ASL contrast shows stable noise characteristics over the entire frequency spectrum, which makes it suitable for studying low-frequency events in brain function. The present study investigates the relative sensitivities of ASL and BOLD contrast in detecting changes in motor cortex activation over a spectrum of frequencies of experimental design, where the alternating period between the resting state and activation is varied from 30 s up to 24 hr. The results demonstrate that 1) ASL contrast can detect differences in motor cortex activation over periods of minutes, hours, and even days; 2) the functional sensitivity of ASL contrast becomes superior to that of BOLD contrast when the alternating period between the resting state and activation is greater than a few minutes; and 3) task activation measured by ASL tends to have less intersubject variability than BOLD contrast. The improved sensitivity of the ASL contrast for low task frequency and longitudinal studies, along with its superior power in group analysis, is expected to extend the range of experimental designs that can be studied using fMRI.

Adult↗
PubMedDate not supplied

[Determination of the correlation time of the lysozyme molecule and lysozyme-inhibitor complex using a spin-label method].

Temperature relationships of rotational correlation times (tau M) of lysozyme molecules were studied using viscosity method based on the model of slow feebly anisotropic rotation of label N-O-group. Protein is mainly associated in 0.01 phosphate buffer (pH 7.3) at 5-30 degrees C. Formation of the complex of lysozyme with competitive inhibitor (3-N-AGA) leads to changes of tau M. It may be due to a decrease of polymerization degree and increase of the protein packing. It is suggested that two-component form of ESR spectra of different spin labeled proteins reflects their common ability for pulsations between more and less compact conformers due to the thermal relative movements of domains and subunits.

Acetylglucosamine↗
PubMed2000

Detection of regional pulmonary perfusion deficit of the occluded lung using arterial spin labeling in magnetic resonance imaging.

Detection of regional perfusion deficit in the lung has been demonstrated using an arterial spin labeling technique called flow-sensitive alternating inversion recovery with an extra radiofrequency pulse (FAIRER). A pulmonary artery was occluded using a nondetachable balloon catheter to simulate an acute pulmonary embolism in 3 of 10 rabbits. Inflating the balloon occludes the artery, and deflating the balloon allows for reperfusion. Perfusion imaging was performed pre-occlusion, during occlusion, and after reperfusion. Signal enhancement due to perfusion of the pulmonary parenchyma was observed in the perfusion images with negligible artifacts. The perfusion deficit of the pulmonary parenchyma was detected distal to the site of occlusion in all three rabbits. Return of the pulmonary parenchymal perfusion was observed after reperfusion. Magnetic resonance imaging using FAIRER can detect signal loss due to absence of perfusion caused by pulmonary embolism.

Animals↗
PubMed1985

Erythrocyte membrane abnormalities in patients with amyotrophic chorea with acanthocytosis. Part 1. Spin labeling studies and lipid analyses.

Low fluidity was observed in the erythrocyte membranes of familial chorea-acanthocytosis by spin labeling with 12NS. The high content of the saturated fatty acids support the finding of low fluidity. Other lipid contents (cholesterol and phospholipids) and the cholesterol/phospholipid ratio were within normal ranges. The sharp decrease in the parameter, (h0/h-1)1/2, suggests phase separation deep inside the patients' erythrocyte membrane matrix. This distortion of the membrane matrix may be caused by membrane components other than the lipid components.

Acanthocytes↗
PubMed2006

Synthesis and biological evaluation of new spin-labeled derivatives of podophyllotoxin.

In order to find compounds with superior bioactivity and less toxicity, a series of spin-labeled podophyllotoxin derivatives were synthesized and tested for the partition coefficients and cytotoxicity against P-388 and A-549. Furthermore, we also determined antioxidant activities of target molecular in tissues of SD rats by the TBA method. Results revealed that most synthesized compounds showed more significant cytotoxicity against P-388 and A-549 in vitro than VP-16. Among them, 9d exhibited most potent cytotoxicity against P-388 and A-549 cells (IC50 is <0.01 and 0.13 microM, respectively). Also, the antioxidative activities showed that the modified compounds of 4'-demethylepipodophyllotoxin (9a-d and 10a-c) are higher than those of podophyllotoxin series (8a-d). The relationship between the cytotoxicity and antioxidative activity discussed.

Animals↗
PubMed2002

Differential effects of cholesterol on acyl chain order in erythrocyte membranes as a function of depth from the surface. An electron paramagnetic resonance (EPR) spin label study.

The purpose of this work is to analyze the effects of cholesterol modulation on acyl chain ordering in the membrane of human erythrocytes as a function of depth from the surface. Partial cholesterol depletion was achieved by incubation of erythrocytes with liposomes containing saturated phospholipids, or with methyl-beta-cyclodextrin (MbetaCD). Cholesterol enrichment was achieved by incubation with liposomes formed by phospholipids/cholesterol, or with the complex MbetaCD/cholesterol. Acyl chain order was studied with electron paramagnetic resonance spectroscopy (EPR) using spin labels that sense the lipid bilayer at different depths. It is shown that the increase in cholesterol stiffens acyl chains but decreases the interaction among lipid headgroups, while cholesterol depletion causes the opposite behavior. It is likely that the observed cholesterol effects are related to those stabilizing the cholesterol-rich detergent-insoluble membrane domains (rafts), recently shown to exist in erythrocytes.

Acylation↗
PubMed2005

Comparison of continuous overt speech fMRI using BOLD and arterial spin labeling.

Overt speech production in functional magnetic resonance imaging (fMRI) studies is often associated with imaging artifacts, attributable to both movement and susceptibility. Various image-processing methods have been proposed to remove these artifacts from the data but none of these methods has been shown to work with continuous overt speech, at least over periods greater than 3 s. In this study natural, continuous, overt sentence production was evaluated in normal volunteers using both arterial spin labeling (ASL) and conventional echoplanar blood oxygenation level-dependent (BOLD) imaging sequences on the same 1.5-T scanner. We found a high congruency between activation results obtained with ASL and the de facto gold standard in overt language production imaging, positron emission tomography (PET). No task-related artifacts were found in the ASL study. However, the BOLD data showed artifacts that appeared as large bilateral false-positive temporopolar activations; percent signal change estimated in these regions showed signal increases and temporal dynamics that were incongruent with typical BOLD activations. These artifacts were not distributed uniformly, but were aligned at the frontotemporal base, close to the oropharynx. The calculated head movement parameters for overt speech blocks were within the range of the rest blocks, indicating that head movement is unlikely the reason for the artifact. We conclude that ASL is not influenced by overt speech artifacts, whereas BOLD showed significant susceptibility artifacts, especially in the opercular and insular regions, where activation would be expected. ASL may prove to be the method of choice for fMRI investigations of continuous overt speech.

Adult↗