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PubMed2002

Antioxidative activity of spin labeled derivatives of podophyllic acid hydrazide.

AIM: To study the relationship between structure and antioxidation activity of spin labeled derivatives of podophyllic acid hydrazide (GP) in tissues and red blood cells (RBC) from rats. METHODS: The homogenate of liver, heart, and kidneys of rats was used to measure malondialdehyde (MDA) spontaneous generated and induced by hydroxyl free radical generation system (Fe2+-ascorbic acid, FRGS) or doxorubicin (DOX) by TBA colorimetric method. H2O2-caused hemolysis was determined spectrophotometrically. Superoxide anion from zymosan-stimulated neutrophils of rats was evaluated by NBT-reduction assay. RESULTS: GP1 and GP1OH obviously inhibited MDA formation either spontaneously or induced by FRGS and DOX and antagonized hemolysis induced by H2O2, but GP and GP1H showed less potent activity. GP1 also inhibited the formation of superoxide anion from activated neutrophils of rats. CONCLUSION: Introduction of nitroxyl radical moieties into GP generated potent derivatives with antioxidative activity. The essential antioxidation active groups of spin labeled derivative of GP are NO or NOH group in nitroxyl radical moieties.

Animals↗
PubMedDate not supplied

[Study of the microstructure of the water-protein layer of spin-labeled preparations of lysozyme by the magnetic relaxation method].

Possible applications of magnetic relaxation to the studies of microstructures of water-protein layer of spin-labelled proteins and enzymes are analyzed on lyzozymes taken as an example. It is shown that the effective time of correlation of dipole-dipole interaction of the complex spin label--water proton (Tauc), as well as thermodynamic parameters Eeff. and delta Seff. reflect the local state of solvatic surrounding of lyzozyme in case of long labels. Conformation transitions of lyzozyme are revealed at the change of temperature (5--80 degrees C) and as a result of binding of a specific inhibitor NAG. It is concluded that local physico-chemical properties of water-protein matrix of lyzozyme spin-labelled preparations depend on protein conformational state.

Chemical Phenomena↗
PubMed1988

Detection of malignant hyperthermia susceptibility using a spin label technique on red blood cells.

Using spin labelled red blood cells and electron paramagnetic resonance spectroscopy (EPR), we observed that halothane 3 mmol litre-1 produced a much greater decrease in the rotational correlation time of red blood cells from individuals who were at risk for malignant hyperthermia (MH), compared with normals. Subsequently we performed blind tests on 14 individuals whose MH susceptibility status had been determined on the basis of in vitro contracture studies. When compared with the results of the in vitro contracture studies, the EPR studies predicted correctly the patients' status in 13 of the 14 individuals.

Cyclic N-Oxides↗
PubMed2004

Brain perfusion territory imaging applying oblique-plane arterial spin labeling with a standard send/receive head coil.

A new method for the selective spin labeling of left- or right-sided supplying arteries of the brain without the need for additional RF coils is demonstrated. A clinical 1.5 T scanner was used. The spatial selectivity of the labeling process is based on the limited coverage of the excitation field of a standard send/receive head coil together with an oblique positioning of the labeling plane. A computer simulation was used to optimize key labeling parameters under the condition of laminar flow. The validity of the computer model results was confirmed by MRI measurements with a flow model. For human studies, a double-inversion continuous arterial spin labeling (CASL) sequence was modified to allow for arbitrary positioning of the labeling plane. The obtained perfusion-weighted images showed a clear delineation of the perfusion territories of the selected arteries in the anterior circulation of the brain and good gray/white matter contrast.

Brain Mapping↗
PubMed1984

Identification of apical membranes from tight epithelia using spin-labeled amiloride and electron paramagnetic resonance spectroscopy.

Apical cell membranes from Na+-transporting epithelia were identified in centrifugal fractions prepared from homogenates of rainbow trout kidney, gill and frog skin using a spin-labeled, nitroxide derivative of amiloride and electron paramagnetic resonance spectroscopy. Spin-labeled amiloride (ASp) is a potent inhibitor of Na+ transport. Frog skin short-circuit current was inhibited by 50% in the presence of 7 X 10(-8) M ASp, whereas 4 X 10(-7) M amiloride was required to obtain the same effect. ASp is a suitable probe for the amiloride binding site based on analytical criteria: Unbound ASp produces an EPR signal linear with concentration and detectable at micromolar concentrations. Estimates of ASp binding can usually be made on less than 100 micrograms of membrane protein. While ASp binds nonspecifically to many materials, amiloride- or benzamil-displaceable binding occurred only in trout gill and kidney, and in frog skin, but not in trout skeletal muscle. ASp binds to membrane fractions produced by differential centrifugation of trout gill, kidney and frog skin. In trout gill and kidney, 81% and 91%, respectively, of the amiloride-displaceable ASp binding is found in the 10,000 X g fraction. All of the ASp binding in frog skin is found in the 10,000 X g fraction. These data indicate that spin-labeled amiloride is a useful probe for the identification of the amiloride binding site, and electron paramagnetic resonance spectroscopy will allow the amiloride binding site to be used as a molecular marker for apical membranes.

Amiloride↗
PubMed2006

Detrimental effects of BOLD signal in arterial spin labeling fMRI at high field strength.

Arterial spin labeling (ASL) MRI is a useful technique for noninvasive measurement of cerebral blood flow (CBF) in humans. High field strength provides a unique advantage for ASL because of longer blood T(1) relaxation times, making this technique a promising quantitative approach for functional brain mapping. However, higher magnetic field also introduces new challenges. Here it is shown that the CBF response determined using ASL functional MRI (fMRI) at 3.0 T contains significant contamination from blood-oxygenation-level-dependent (BOLD) effects. Due to interleaved acquisitions of label and control images, difference in blood oxygenation status between these two scans can cause incomplete cancellation of the static signal upon image subtraction, resulting in a BOLD-related artifact in the estimated CBF hemodynamics. If not accounted for, such an effect can complicate the interpretation of the ASL results, e.g., causing a delayed onset and offset of the response, or inducing an artifactual poststimulus undershoot. The BOLD contribution also decreases the sensitivity of ASL-based fMRI. Correction methods are proposed to reduce the artifact, giving increased number of activated voxels (18+/-5%, P=0.006) and more accurate estimation of CBF temporal characteristics.

Algorithms↗
PubMed2004

Spin-labeled gramicidin a: channel formation and dissociation.

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.

Electron Spin Resonance Spectroscopy↗
PubMed1992

Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation.

A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).

Adenine Nucleotides↗
PubMed1988

Rotational diffusion of a steroid molecule in phosphatidylcholine membranes: effects of alkyl chain length, unsaturation, and cholesterol as studied by a spin-label method.

Rotational diffusion of cholestane spin-label (CSL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of chain length and unsaturation of alkyl chains, cholesterol mole fraction, and temperature for better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. CSL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines are obtained for various membranes in the liquid-crystalline phase. The wobbling diffusion constant decreases in the order dilauroyl-PC greater than dimyristoyl-PC greater than dioleoyl-PC approximately dipalmitoyl-PC greater than distearoyl-PC greater than dioleoyl-PC/cholesterol = 3/1 greater than dioleoyl-PC/cholesterol = 1/1 membranes. Activation energy for the wobbling diffusion of the long axis of CSL is strongly dependent on alkyl chain length, unsaturation, and cholesterol mole fraction. It decreases with decrease in alkyl chain length and by introduction of unsaturation in the alkyl chains. In dioleoylphosphatidylcholine membranes, activation energy decreases by a factor of approximately 3 in the presence of 50 mol % cholesterol. Activation energy for wobbling diffusion of CSL in phosphatidylcholine membranes is smaller than the activation energy for translational diffusion of a phospholipid. The former is more dependent on alkyl chain length and unsaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanes↗
PubMed2000

Magnetic resonance perfusion imaging in acute ischemic stroke using continuous arterial spin labeling.

BACKGROUND AND PURPOSE: Continuous arterial spin-labeled perfusion MRI (CASL-PI) uses electromagnetically labeled arterial blood water as a diffusible tracer to noninvasively measure cerebral blood flow (CBF). We hypothesized that CASL-PI could detect perfusion deficits and perfusion/diffusion mismatches and predict outcome in acute ischemic stroke. METHODS: We studied 15 patients with acute ischemic stroke within 24 hours of symptom onset. With the use of a 6-minute imaging protocol, CASL-PI was measured at 1.5 T in 8-mm contiguous supratentorial slices with a 3.75-mm in-plane resolution. Diffusion-weighted images were also obtained. Visual inspection for perfusion deficits, perfusion/diffusion mismatches, and effects of delayed arterial transit was performed. CBF in predetermined vascular territories was quantified by transformation into Talairach space. Regional CBF values were correlated with National Institutes of Health Stroke Scale (NIHSS) score on admission and Rankin Scale (RS) score at 30 days. RESULTS: Interpretable CASL-PI images were obtained in all patients. Perfusion deficits were consistent with symptoms and/or diffusion-weighted imaging abnormalities. Eleven patients had hypoperfusion, 3 had normal perfusion, and 1 had relative hyperperfusion. Perfusion/diffusion mismatches were present in 8 patients. Delayed arterial transit effect was present in 7 patients; serial imaging in 2 of them showed that the delayed arterial transit area did not succumb to infarction. CBF in the affected hemisphere correlated with NIHSS and RS scores (P=0.037 and P=0.003, Spearman rank correlation). The interhemispheric percent difference in middle cerebral artery CBF correlated with NIHSS and RS scores (P=0.007 and P=0.0002, respectively). CONCLUSIONS: CASL-PI provides rapid noninvasive multislice imaging in acute ischemic stroke. It depicts perfusion deficits and perfusion/diffusion mismatches and quantifies regional CBF. CASL-PI CBF asymmetries correlate with severity and outcome. Delayed arterial transit effects may indicate collateral flow.

Acute Disease↗
PubMed1994

1H NMR and spin-labeled EPR studies on the interaction of calmodulin with jujuboside A.

Jujuboside A (JuA), an effective component of sanzaoren, a Chinese herbal medicine, is a noncompetitive inhibitor of calmodulin (CaM). The interaction of JuA with CaM has been investigated with 1H NMR and spin-labeled EPR spectroscopies. The 1H NMR experiments showed that JuA has two kinds of binding sites on CaM: one locates in the N-terminus, the other locates in the C-terminal region of the polypeptide chain. The EPR studies on 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl labeled CaM (spin-labeled CaM) revealed that each CaM molecule can bind at least two JuA molecules. Binding of JuA affects the environments of some lysine residues (most likely Lys-74 and Lys-94), suggesting that JuA binds to CaM through hydrophobic interaction.

Amino Acid Sequence↗
PubMed1999

Nitroxide side-chain dynamics in a spin-labeled helix-forming peptide revealed by high-frequency (139.5-GHz) EPR spectroscopy.

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

Amino Acid Sequence↗
PubMed1995

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers.

We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.

Animals↗
PubMed1973

Orientation and motion of amphiphilic spin labels in hexagonal lipid phases.

The acyl chain of spin-labeled fatty acids intercalates between the lipid hydrocarbon chains in hexagonal and micellar phases with the carboxyl group anchored at the lipid water interface. The spectra are characteristic of anisotropic motion and cannot be distinguished from the spectra of these probes in lamellar dispersions. In the hexagonal and micellar phases the molecular motion of the spin label increases as it is moved further away from the carboxyl group, similar to the behavior in the lamellar phase (Jost et al. (1971) J. Mol. Biol. 59, 77-98). The similarity in packing of the acyl chains in the hexagonal and lamellar phases suggests that localized regions of hexagonal phase are compatible with a bilayer matrix.

Calcium↗
PubMed1983

[Preparation and ESR study of spin-labeled derivatives of Naja naja oxiana neurotoxin II].

After neurotoxin II Naja naja oxiana reaction with N-hydroxysuccinimidyl 2,2,6,6-tetramethyl-4-carboxymethylpiperidine-1-oxyl, six derivatives were isolated, each containing one spin label. Their analysis (reduction, carboxymethylation, tryptic hydrolysis, isolation and identification of the spin labeled peptide) allowed to localize the label position: the epsilon-amino groups of Lys15, Lys25, Lys26, Lys44, Lys48, and alpha-amino group of Leu1. The neurotoxin II reaction with N-hydroxysuccinimidyl 2,2,5,5-tetramethyl-3-carboxypyrrolin-1-oxyl followed by chromatography afforded 10 derivatives, each having two labeled lysine residues, wherein the position of the modified residues was determined. The reactivity and microenvironment of amino groups are discussed basing on the dependence between the reaction conditions and yields. For di-spin labeled derivatives of the pyrroline series, the inter-label distances were determined by EPR from the standard curve and used for refinement of the neurotoxin conformation in solution.

Amino Acid Sequence↗
PubMed1975

ESR study of aqueous dispersions of beta-lactoglobulin and spin-labelled glyceryl monostearate.

From the ESR spectra of aqueous dispersions of synthetic glyceryl monostearate (spin labelled at C-12) a critical micelle concentration of 30 mumol/l at room temperature was obtained, which agrees with that deduced from surface tension measurements. At monoglyceride concentrations smaller or larger than the critical micelle concentration, the monomers show increased motional restriction with increasing molar ratio of beta-lactoglobulin to monoglyceride up to a value of 10, as determined from calculated rotational correlation times; A similar progressive interaction was deduced from spectral changes observed on equimolar dispersions of beta-lactoglobulin and monoglyceride on raising the temperature to 55 degrees C at which the protein and monoglyceride coprecipitate. The relevance of these finding for non-labelled monoglyceride dispersions is indicated by the similarity of the pH-dependent flocculation behaviour of labelled and non-labelled monoglycerides, both in the absence and presence of beta-lactoglobulin; In addition, proton magnetic resonance and mechanical stability measurements suggest that spin-labelled glyceryl monosterate behaves analogously to non-labelled glyceryl monooleate.

Binding Sites↗
PubMed1991

Spin labeling studies of wheat germ calmodulin in solution.

Electron paramagnetic resonance was used to investigate the physical state of plant calmodulin in solution. Wheat germ calmodulin contains a single cysteine residue (Cys-27) on the first of four calcium binding loops. In this study the nitroxide spin label 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl (MAL-6) was covalently attached to Cys-27 to produce a Ca(2+)-sensitive, biologically-active, labeled protein. The rotational correlation time of the spin label, a measure of its rotational mobility and reflective of the physical state of this region of the protein, was calculated under various conditions. Relative to control, changes in the physical state of the protein reflected by increased motion of the spin label were observed at high pH, low ionic strength and upon addition of Ca2+. These results extend knowledge of the structure of the protein, previously known from solid state and biochemical studies, to calmodulin in solution.

Calmodulin↗
PubMed2006

Discovering new drug-targeting sites on flexible multidomain protein kinases: Combining segmental isotopic and site-directed spin labeling for nuclear magnetic resonance detection of interfacial clefts.

A novel structure-based approach to study the structure and dynamics of flexible multi-domain monomeric protein kinases, which otherwise do not yield diffraction quality crystals, is described. A combination of segmental 15N-isotopic labeling of a regulatory domain with site-directed paramagnetic nitroxide spin labeling of the kinase domain is employed. Nuclear magnetic resonance studies of the enhancement of amide proton relaxation rates of the 15N-isotopically labeled regulatory domain caused by insertion of the paramagnetic nitroxide spin label on the kinase domain provide long-range distance restraints for determination of both the average positional structure and the relative flexibility exhibited between the two contiguous domains. Clefts and crevices detected around the dynamic domain-domain interface provide new targeting sites for tethered-based extension of current small-molecule lead compounds to produce more potent and selective pharmaceutical agents.

Drug Delivery Systems↗