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PubMed1983

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.

Electron Spin Resonance Spectroscopy↗
PubMed2004

Distinguishing between anterior cerebral artery and middle cerebral artery perfusion by color-coded perfusion direction mapping with arterial spin labeling.

The purpose of this study was to evaluate collateral circulation by describing anterior cerebral artery and middle cerebral artery perfusion areas. Pairs of image sets spin labeled on the medial and lateral side were used. A pixel-by-pixel t test was performed, with blue gradation used to display lateral perfusion (ie, middle cerebral artery) and orange gradation for anterior cerebral artery perfusion. Extensions of anterior cerebral artery perfusion areas in cases of middle cerebral artery stenosis were described. This method may aid in estimation of collateral circulation for stroke treatment.

Cerebral Angiography↗
PubMed1993

Sarcoplasmic reticulum calcium ATPase. Labeling of a putative Mg2+ site by reaction with a carbodiimide and a spin-label.

The sarcoplasmic reticulum Ca(2+)-ATPase loses hydrolytic activity and the ability to be phosphorylated by Pi following incubation with EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. 4 nmol of tempamine per mg SR protein can be coupled to either a glu or an asp side chain through the EDC reaction. Mg2+ protects against loss of activity and tempamine labeling with a mid-point of about 3 mM in the absence of Ca2+. This is similar to the Kd for a Mg2+ that serves as a cofactor in enzyme phosphorylation. The Mg2+ protection constant is lowered by an order of magnitude when Ca2+ is bound to the transport sites. It is suggested that control of the Mg2+ binding site affinity may be part of the mechanism of enzyme activation by Ca2+.

Animals↗
PubMed2005

Multislice continuous arterial spin-labeled perfusion MRI in patients with chronic occlusive cerebrovascular disease: a correlative study with CO2 PET validation.

PURPOSE: To compare the use of multislice continuous arterial spin labeling (CASL) and CO(2) positron emission tomography (PET) to assess CBF in patients with chronic occlusive cerebrovascular disease for the validation of quantitative CASL perfusion in an altered hemodynamic state. MATERIALS AND METHODS: Eleven patients with occlusive carotid artery disease were studied with CASL and conventional MRI. Cerebral blood flow (CBF) was also measured with O(15)-labeled CO(2) gas using a whole-body PET scanner. The average values within region of interests (ROIs) drawn on coregistered CASL and PET images were used for the linear regression analysis and to assess the effect of transit time on the quantification using CASL. RESULT: In all patients there was a significant correlation between the CBF values from CASL and PET (r = 0.71 +/- 0.07, P < 0.001). The slope of regression lines varied widely among patients (0.54-1.77). Longer transit times were estimated in four of 11 patients in the hypoperfusion region. CONCLUSION: Quantification of CBF using CASL is feasible and reasonable, even when employed in a routine clinical setting. However, the long transit time may lead to underestimation of the affected side in occlusive cerebrovascular disease.

Aged↗
PubMed1986

Association of a spin-labeled local anesthetic with the allosterically coupled noncompetitive inhibitor site on the acetylcholine receptor.

Radioligand binding and ESR were used to study the association of a spin-labeled local anesthetic, 2-[N,N-dimethyl-N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-4-hexyloxybenzoate iodide (C6SLMel), with acetylcholine receptor-enriched membranes from Torpedo californica. In the presence of carbamylcholine, we found that C6SLMel competitively inhibits [3H]phencyclidine binding with high affinity (KD = 8.7 X 10(-7) M for C6SLMel), whereas in the presence of alpha-toxin or in the absence of agonist, C6SLMel binds with lower affinity (KD = 2 X 10(-5) M). At concentrations lower than 1 X 10(-5) M, C6SLMel does not bind to the agonist site but enhances [3H]acetylcholine binding. These findings show that C6SLMel binds to the allosterically coupled noncompetitive inhibitor site which is regulated by agonist binding. In addition, C6SLMel preferentially associates with the desensitized receptor state known to exhibit high affinities for agonists and local anesthetics. ESR measurements of C6SLMel bound to receptor membranes in the absence of agonist show moderately immobilized spectra. Addition of carbamylcholine results in the appearance of a strongly immobilized component. Prior exposure to alpha-toxin blocks the carbamylcholine-induced, strongly immobilized component in the ESR spectrum. Furthermore, in the presence of carbamylcholine, back-titration of bound C6SLMel with phencyclidine decreases the highly immobilized component at concentrations consistent with the KD for phencyclidine. These findings indicate that C6SLMel detects conformational changes between the resting and desensitized acetylcholine receptor states that occur at the noncompetitive inhibitor binding site. The strongly mobilized component is not affected by ferricyanide addition, suggesting that the binding site is in a region not readily accessible to anion collision from the aqueous phase.

Acetylcholine↗
PubMed2003

Selective arterial spin labeling (SASL): perfusion territory mapping of selected feeding arteries tagged using two-dimensional radiofrequency pulses.

To date, most perfusion magnetic resonance imaging (MRI) methods using arterial spin labeling (ASL) have employed slab-selective inversion pulses or continuous labeling within a plane in order to obtain maps derived from all major blood vessels entering the brain. However, there is great potential for gaining additional information on the territories perfused by the major vessels if individual feeding arteries could be tagged. This study demonstrates noninvasive arterial perfusion territory maps obtained using two-dimensional (2D) selective inversion pulses. This method is designated "selective ASL" (SASL). The SASL method was used to tag the major arteries below the circle of Willis. A combination of 2D selective tagging and multislice readout allows perfusion territories to be clearly visualized, with likely applications to cerebrovascular disease and stroke.

Brain↗
PubMedDate not supplied

[Spin-labeling studies on interaction of phospholipase A2 from cobra poison with phospholipid membranes].

Phospholipase A2 interactions with phospholipid liposomes and vesicles were studied by using unhydrolysed spin-labeling 2-alkyl phosphatidylcholine. These interactions have polar nature. Phospholipase A2 hydrolyses only the neighbouring phospholipid monolayer. The hydrophobic layer of liposomes is not permeable for the enzyme. Phospholipase A2 are attached to lysophosphatidylcholine after hydrolysis and it stabilizes the structure of vesicles. The stabilizing effect of the enzyme makes the vesicles unpermeable for Na+, Cl-ions and protons. Moreover, the phospholipase A2 prevents the phospholipid exchange between the vesicles and their fusion.

Animals↗
PubMed1984

Binding of a spin-labelled photoallergen to human serum albumin.

The binding site for 3,3',4',5-tetrachlorosalicylanilide (T4CS), a potent photoallergen, on human serum albumin (HSA) was studied by electron spin resonance spectroscopy using a spin-labelled analogue 3,5-dichlorosalicylamido-4-(2,2,6,6-tetramethylpiperidine 1-oxyl) (DCS-TEMPO) of T4CS in the absence of ultraviolet irradiation. DCS-TEMPO bound non-covalently (K = 5.8 X 10(6) M-1) to one major binding site on HSA. This binding site could be blocked by the photochemical binding of T4CS to the protein. Limited tryptic digestion of HSA or chemical modification of its single tryptophan residue with 2-hydroxy-5-nitrobenzyl bromide was found to reduce the binding constant of the T4CS/DCS-TEMPO-binding site. These observations are in good agreement with earlier conclusions on the nature of the T4CS-binding site and suggest a location for this site close to the single tryptophan residue of the HSA molecule.

Allergens↗
PubMed2006

Quantitative perfusion imaging using arterial spin labeling.

MRI-based perfusion imaging techniques can be classified into those that use exogenously administered contrast agents and those that use an endogenous material that reflects blood flow. This chapter focuses on the technique of arterial spin labeling (ASL), in which endogenous water is made a freely diffusible perfusion tracer by perturbing the magnetization of blood water in arteries prior to their entry into tissue of interest. The technique is totally noninvasive and allows repeated quantitative blood flow measurements in a time scale limited only by the spin lattice relaxation time (T1). Absolute quantitation requires measurement of T1, transit time, and labeling efficiency, as well as careful control for magnetization transfer effects. Two main variants of the ASL technique are in use: continuous ASL (CASL) and pulsed ASL (PASL). This chapter describes basic theory for CASL, and experimental and computational procedures for obtaining quantitative perfusion maps of the brain. Extension of the technique for renal perfusion imaging is outlined.

Animals↗
PubMedDate not supplied

[A study of thermoinduced changes in human high-density lipoproteins in hypo-alpha-lipoproteinemia and ischemic heart disease by the spin label method].

Thermo-induced structural reorganization in human high density lipoproteins subclasses HDL2 and HDL3 have been studied by means of electronic paramagnetic resonance method. A number of spin labels were used: 5- and 16-DOXYL stearates, DOXYL-labelled derivatives of pentadecane, phosphatidyl choline and sphingomyelin. In hypoalphalipoproteinemia one of the thermo-induced transitions in HDL2 (at 23-30 degrees) was not observed. In coronary heart disease the critical temperature for HDL3 was shifted from the physiological values and located at 42-48 degrees. The data obtained are discussed from the point of view of their potential role in atherogenesis.

Adult↗
PubMed1999

Spatial and temporal modulation of perfusion in the rat ovary measured by arterial spin labeling MRI.

The hemodynamic changes triggered by luteinizing hormone/human chorionic gonadotropin (LH/hCG) in ovaries of immature pregnant mare serum gonadotropins (PMSG)-primed female Wistar rats were followed by pulsed arterial spin labeling magnetic resonance imaging. Decreased perfusion was monitored in the first 2 hours after administration of hCG followed by a transient significant rise in perfusion. Subsequently, constant ovarian perfusion of 10.9 +/- 4.3 mL min(-1) g(-1) was maintained during the exponential increase in ovarian volume. However, ovarian perfusion was not uniform, and prior to ovulation poorly perfused regions were detected that were assigned to the follicular fluid in preovulatory follicles. This result implied that in the time scale of seconds, corresponding to the T1 relaxation time of water in the follicular fluid, exchange of arterial water with water in the follicular fluid was negligible. Along with the drop in the levels of high-energy phosphate metabolites detected by 31P nuclear magnetic resonance spectroscopy and the shift to glycolytic metabolism, these results support the hypothesis that physiological hypoxia could play a role in large preovulatory follicles as part of the normal ovarian cycle.

Animals↗
PubMed1984

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody.

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.

Antibodies, Monoclonal↗
PubMed1982

Ethanol disordering of spin-labeled mouse brain membranes: correlation with genetically determined ethanol sensitivity of mice.

Disordering of brain and erythrocyte membranes by ethanol in vitro was measured by ESR using 5-doxylstearic acid as spin label. Synaptosomal plasma membranes and erythrocyte membranes were isolated from two lines of mice developed, by selective breeding, for differential sensitivity to hypnotic effects of ethanol. Membranes taken from alcohol-sensitive "long-sleep" mice were more strongly disordered by ethanol in vitro than were membranes from alcohol-resistant "short-sleep" mice. Furthermore, within a population of genetically heterogeneous mice, the most ethanol-sensitive animals had the most ethanol-sensitive synaptosomal plasma membranes. In vivo sensitivity of the individual mice was evaluated by measuring brain ethanol levels at a precise behavioral end point, recovery from ataxia. The data extend our previous observations of correlations between in vitro and in vivo effects of ethanol and suggest that membrane disordering may be a primary mechanism of acute effects of ethanol.

Animals↗
PubMed1983

Electron spin resonance spin label studies of the erythrocyte membrane in Duchenne muscular dystrophy.

The membrane organisation of erythrocytes from patients with Duchenne muscular dystrophy (DMD) and from asymptomatic carriers was studied by the electron spin resonance (ESR) spin label technique. Following the work of Sato et al [1978], we used 2-(14-carboxytetradecyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyloxyl as probe. We found no significant difference in lipid fluidity at 30 degrees C, measured by the ratio of the line height at central field to that at high field ho/h-1, in DMD patients or carriers compared to appropriate control persons. Our conclusions for the dystrophic boys differ from those of Sato et al [1978], although our data are consistent with theirs within experimental error. We also studied the variation of the ratio ho/h-1 over the temperature range 5-35 degrees C in these individuals. For normal erythrocytes there is a discontinuity in this plot around 15 degrees C that is absent in the erythrocytes of DMD patients [in agreement with the findings of Sato et al, 1978] and also absent in DMD carriers. We suggest the slope of this logarithmic temperature plot over the range 15-35 degrees C is a more useful empirical parameter as the presence of these discontinuities is sometimes uncertain, and using this parameter, we find a clear separation between carriers and controls.

Adult↗
PubMed2003

Structural organization of alpha-synuclein fibrils studied by site-directed spin labeling.

Despite its importance in Parkinson's disease, a detailed understanding of the structure and mechanism of alpha-synuclein fibril formation remains elusive. In this study, we used site-directed spin labeling and electron paramagnetic resonance spectroscopy to study the structural features of monomeric and fibrillar alpha-synuclein. Our results indicate that monomeric alpha-synuclein, in solution, has a highly dynamic structure, in agreement with the notion that alpha-synuclein is a natively unfolded protein. In contrast, fibrillar aggregates of alpha-synuclein exhibit a distinct domain organization. Our data identify a highly ordered and specifically folded central core region of approximately 70 amino acids, whereas the N terminus is structurally more heterogeneous and the C terminus ( approximately 40 amino acids) is completely unfolded. Interestingly, the central core region of alpha-synuclein exhibits several features reminiscent of those observed in the core region of fibrillar Alzheimer's amyloid beta peptide, including an in-register parallel structure. Although the lengths of the respective core regions differ, fibrils from different amyloid proteins nevertheless appear to be able to take up highly similar, and possibly conserved, structures.

Amyloid beta-Peptides↗
PubMed1983

A spin label study on the effect of dibutyryl cyclic AMP on synaptic plasma membranes isolated from rat brain.

The fluidity of the deep layer of the lipid bilayer and the mobility of sulphydryl (-SH) groups of synaptic plasma membranes, isolated from rat brain and spin-labelled with a doxyl derivative of stearic acid and with a nitroxide derivative of maleimide, respectively, were studied using electron spin resonance (ESR) techniques. Dibutyryl cyclic AMP (db-cAMP) and adenosine triphosphate (ATP) had no effect on the fluidity of the deep layer of the lipid bilayer of synaptic plasma membranes. While protein dephosphorylation was inhibited by Zn2+, db-cAMP together with ATP increased the mobility of -SH groups above the value obtained with db-cAMP alone.

Adenosine Triphosphate↗
PubMed1985

Interaction of vinblastine sulfate with artificial phospholipid membranes. A study by differential scanning calorimetry and spin labeling.

The effect of the antimitotic drug vinblastine sulfate has been studied on fully hydrated dipalmitoylphosphatidylcholine (DPPC) liposomes in the temperature range 0 degrees to 60 degrees using differential scanning calorimetry and electron spin resonance spectroscopy with two fatty acid spin labels. In the gel phase, vinblastine interacts essentially with the DPPC polar heads and induces an important disorganization of the phospholipidic bilayer. The co-operativity of the main thermal transition is decreased. In the crystal-liquid phase, the drug penetrates inside the artificial membrane and induces the formation of domains which increased thermal stability. These effects are opposite to those observed with the drug isaxonine which is used to reduce the axonal degenerating effects due to vinblastine.

Binding Sites↗
PubMed2003

Comparison of arterial spin-labeling techniques and dynamic susceptibility-weighted contrast-enhanced MRI in perfusion imaging of normal brain tissue.

OBJECTIVES: To evaluate relative cerebral blood flow (rCBF) in normal brain tissue using arterial spin-labeling (ASL) methods and first-pass dynamic susceptibility-weighted contrast-enhanced (DSC) magnetic resonance imaging (MRI). METHODS: Sixty-two patients with brain metastases were examined on a 1.5 T-system up to 6 times during routine follow-up after stereotactic radiosurgery. Perfusion values in normal gray and white matter were measured using the ASL techniques ITS-FAIR in 38 patients, Q2TIPS in 62 patients, and the first-pass DSC echo-planar (EPI) MRI after bolus administration of gadopentetate dimeglumine in 42 patients. Precision of the ASL sequences was tested in follow-up examinations in 10 healthy volunteers. RESULTS: Perfusion values in normal brain tissue obtained by all sequences correlated well by calculating Pearson's correlation coefficients (P < 0.0001) and remained unchanged after stereotactic radiosurgery as shown by analysis of variance (P > 0.05). CONCLUSION: Both ASL and DSC EPI MRI yield highly comparable perfusion values in normal brain tissue.

Adult↗