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At least 685 records · Page 38Linked to original sources
PubMed2000

Transit time, trailing time, and cerebral blood flow during brain activation: measurement using multislice, pulsed spin-labeling perfusion imaging.

Transit time and trailing time in pulsed spin-labeling perfusion imaging are likely to be modulated by local blood flow changes, such as those accompanying brain activation. The majority of transit/trailing time is due to the passage of the tagged blood bolus through the arteriole/capillary regions, because of lower blood flow velocity in these regions. Changes of transit/trailing time during activation could affect the quantification of CBF in functional neuroimaging studies, and are therefore important to characterize. In this work, the measurement of transit and trailing times and CBF during sensorimotor activation using multislice perfusion imaging with pulsed arterial spin-labeling is described. While CBF elevated dramatically ( thick similar80.7%) during the sensorimotor activation, sizable reductions of transit time ( thick similar0.11 sec) and trailing time ( thick similar0.26 sec) were observed. Transit and trailing times were dependent on the distances from the leading and trailing edges of the tagged blood bolus to the location of the imaging slices. The effects of transit/trailing time changes on CBF quantification during brain activation were analyzed by simulation studies. Significant errors can be caused in the estimation of CBF if such changes of transit/trailing time are not taken into account.

Brain↗
PubMed1970

Membrane structure: spin labeling and freeze etching of Mycoplasma laidawii.

A spin-labeled fatty acid was incorporated in vivo into the polar lipids of Mycoplasma laidlawii membranes. The electron paramagnetic resonance signal from either intact cells or their extracted lipids reflected the fatty acid composition of the Mycoplasma membranes. Comparison of signals from intact cells, gramicidin-treated cells, heat-treated cells, and extracted lipids indicates that a major portion of the membrane lipids is in a semiviscous hydrocarbon environment. The results also show that the spin label in the intact membrane is slightly but significantly less mobile than it is in protein-free lipid extracts made from these membranes. Correlated electron microscope examinations using the freeze-etch technique reveal particulate components in the hydrophobic region of the membrane. The mobility of the lipids in the intact cell membrane may be influenced by their association with these particles.

Cell Membrane↗
PubMed1993

Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.

Amino Acid Sequence↗
PubMed1996

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach.

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

Adenosine Triphosphate↗
PubMed1980

[Spin label study of slow molecular rotations of globular proteins using microwave saturation effects in electron paramagnetic resonance].

Viscosity, temperature and ionic strength dependences of ESR microwave saturation parameters of spin labelled human oxyhemoglobin (Hb) and bovine serum albumin (BSA) have been studied. The piperidine and pyrrolidine nitroxyl derivatives of maleimide were used as covalent SH reagents for Hb and BSA and the same two derivatives of gamma-benzocarboline and spin labelled stearic acid were used as noncovalent spin probes for BSA. The effects of label binding tightness on ESR spectral parameters were considered. The rotational correlation times were determined using viscosity dependences of the separation of the outer hyperfine extrema and Stokes extrapolations at high viscosities. The ESR microwave saturation parameters of the spin labels were shown to depend just weakly on temperature (at constant eta/t) over the range 0-25 degrees and on g, A values but to be sensitive to protein rotational correlation times up to 10(-4) sec and also to the rotational anisotropy and to the relative motion of the spin label.

Animals↗
PubMed1992

An EPR investigation of spin-labelled erythrocytes as a diagnostic technique for malignant hyperthermia.

Spin labelled (7-, 12- and 16- doxylsteric (DS) acid) erythrocyte (red blood cell) membranes isolated from six malignant hyperthermia susceptible (MHS) and six malignant hyperthermia normal (MHN) volunteer donors were characterized using the order parameter (S) and rotational correlation time (tau r) determined from 37 degrees C, 9 GHz electron paramagnetic resonance spectra. These parameters were found to decrease due to the increasing fluidization of the membrane as the halothane and benzyl alcohol sample concentration is increased and that significant differences exist in the values of S and tau r between the MHS and MHN sample groups, but that the differences in these parameters between the presence and absence of an external fluidizing agent are not significant for both sample groups. In the absence of these fluidizing agents, the mean values and standard errors of S at 37 degrees C for MHS and MHN are 0.643(2) and 0.652(3) for 7-DS, 0.554(2) and 0.563(3) for 12-DS, respectively, and of tau r are 2.139(12) and 2.223(13) ns for 16-DS, respectively. The values of S and tau r are significantly smaller for the MHS group than for the MHN group contrary to some previous reports. These observations revise and extend previous reports and show that the observed values of S and tau r in the absence of a fluidizing agent depend on the erythrocyte membrane structure in MHS and MHN subjects. They also suggest that the potential to use spin-labelled red blood cells as a screening protocol for MH deserves further investigation.

Benzyl Alcohol↗
PubMed1991

Use of paramagnetic chelated metal derivatives of polysaccharides and spin-labeled polysaccharides as contrast agents in magnetic resonance imaging.

Soluble and insoluble polysaccharides were derivatized with diethylenetriaminepentaacetic acid (DTPA) and/or spin-labeled with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). Polysaccharides derivatized with DTPA were prepared via cyanogen bromide activation, coupling to a diamine linker, and to DTPA anhydride. Spin-labeled polysaccharides were also prepared via cyanogen bromide activation. The extent of derivatization for dextran (18 kDa) was about 120 glucose units per DTPA, and for cellulose and starch about 15-30 units per DTPA. For spin-labeled polysaccharides, the average loading ranged from 1 nitroxide per 16 glucose units for starch to 181 for dextran (82 kDa). These derivatized paramagnetic polysaccharides were shown to be more effective relaxants than the small paramagnetic molecules alone. Both soluble and insoluble polysaccharide-linker-DTPA-Gd(III) complexes were effectively cleared from the body (rats) after oral administration. After intravenous administration, the biodistribution of dextran-linker-DTPA-Gd(III) complexes differed significantly from that of GdDTPA. Reduction of the nitroxide by ascorbic acid was retarded in the polysaccharide derivatives, particularly in starch derivatized with both nitroxide and linker-DTPA-Cu(II). These agents showed contrast enhancement in the gastrointestinal tract of rabbits.

Animals↗
PubMed1984

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length.

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.

Animals↗
PubMed2006

Loading and mobility of spin-labeled insulin in physiologically responsive complexation hydrogels intended for oral administration.

Poly(methacrylic acid-g-ethylene glycol) copolymers are pH-responsive complexation hydrogels that have shown promise in in vitro and in vivo results as oral insulin delivery carriers. With the aim of gaining more detailed insight into their performance to further improve the carriers, we spin-labeled insulin and used electron spin resonance (ESR) spectroscopy to follow the loading of the spin-labeled insulin into the copolymer microparticles. A flow through system was developed to monitor continuously and non-invasively the dynamics of the spin-labeled insulin and its surrounding microviscosity during release. Using these methods, the loading efficiency of insulin was determined and was found to match previous HPLC measurements. Additionally, the protein-friendly nature of the hydrogels was demonstrated. The monitoring of the dynamics during flow through provided a rationalization for the unwanted initial burst release in an acidic environment. These studies will aid in the optimization of the system, and will be a basis for subsequent in vivo ESR investigations.

Administration, Oral↗
PubMed1991

Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.

Actins↗
PubMed1988

Solution of the nitroxide spin-label spectral overlap problem using pulse electron spin resonance.

Short-pulse saturation-recovery (SR) electron spin resonance (ESR) methods have been used to measure the lateral diffusion of a nitroxide-labeled cholesterol analogue (3-spiro-[2'-(N-oxyl-4',4'-dimethyloxazoladine)]-cholestane, CSL) in multilamellar liposomal dispersions. SR experiments were performed on samples containing 14NCSL:15NCSL pairs, and recovery signals were analyzed for initial conditions and multiexponential time constants by computer simulation. Rate equations describing the system were written and solved. The time constants contain combinations of electron spin lattice relaxation times Tle for both isotopes and the Heisenberg exchange rate constant Kx. We have investigated the complication that occurs from overlap of ESR spectral fragments from 14N and 15N moieties. The time constants of the multiexponential signals are independent of ESR line shape and position. From Kx, lateral diffusion constants of CSL in dimyristoylphosphatidylcholine (DMPC) were calculated (D = 1.7 x 10(-8) at 27 degrees C and 2.7 x 10(-8) cm2/s at 37 degrees C). It is shown that short-pulse saturation-recovery methods are able to overcome the ESR spectral overlap problem that is encountered in conventional ESR and continuous wave electron-electron double resonance (CW ELDOR) studies of spin-spin interactions. The present method can be extended to more complex situations involving spin labels in different environments with physical and chemical exchange.

Cyclic N-Oxides↗
PubMed1976

Spin labels as probes for tetraphenylboron ion interaction with liposomes.

The effects of tetraphenylboron (TFB) on the molecular organization of lipids within phosphatidylcholine (PC) liposomes were investigated using the spin-labeled method. Perturbations at the surface of the lipid were probed using stearamide and cholestane spin labels; perturbations in the hydrophobic-portion were probed with spin-labeled amphiphilic fatty esters.

Binding Sites↗
PubMed1981

A spin-label study of protein-lipid interaction in sarcoplasmic reticulum of rabbit skeletal muscle.

Whether or not the thermotropic change at about 18 degrees C in the physical state of Ca2+-ATPase protein molecules of sarcoplasmic reticulum membranes could be transmitted to lipids through protein-lipid interactions was investigated using a spin-label technique. Fatty acid spin labels were used to probe the bulk membrane lipids while long-chain spin labels attached at one end to the Ca2+-ATPase molecules through a covalent bond were used to monitor the boundary lipids. The results on the temperature-dependence of alkyl-chain flexibility of lipid molecules indicate that the change in the state of the protein molecules is accompanied by one of the boundary lipids, but not of the bulk lipids.

Animals↗
PubMed1996

Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition.

Electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASL's) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASL's lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9- > 12- > 5- > 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8 degrees C and the cooperative unit to 1/5 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase.

Calorimetry, Differential Scanning↗
PubMed1998

Sterically stabilized liposomes of DPPC/DPPE-PEG:2000. A spin label ESR and spectrophotometric study.

The chain dynamics and the thermotropic phase behavior of sterically stabilized liposomes obtained introducing in the host bilayer matrix of DPPC up to 7 mol% of the polymer-lipid DPPE-PEG:2000 were investigated by spin label electron spin resonance spectroscopy and spectrophotometry. The experimental data indicate that the dispersions have the dynamic and thermotropic characteristics of normal lamellar phase. Moreover, using spin labels that locate both in the interfacial and in the hydrocarbon regions, namely TEMPO-stearate, 5- and 16-PCSL, we find that relative to the unmodified DPPC bilayers, the polymer-grafted bilayers are loosely packed in the interfacial region and have reduced chain mobility in the gel phase. From the temperature dependence of the partition coefficient (P(c)), of the spin probe DTBN between the aqueous and the fluid hydrophobic regions of the bilayers and from the melting curves of the absorbance at 400 nm, we observe a slight influence on the endothermic phase transitions when increasing the concentration of the polymer-lipid in the DPPC bilayers, the influence being more evident in the pre-transition.

Journal Article↗
PubMed1985

Comparative study of the lipid dynamics in the surface layer of porcine and human high density lipoprotein subclasses by spin labeling.

In order to obtain information on the determinants of the lipid dynamics in the high density lipoproteins (HDL), we have compared the structural properties of human HDL subclasses with porcine HDL artificially subdivided into density subfractions corresponding to those of human HDL. Four different positional isomers of spin labeled fatty acids and spin labeled androstanol experienced more restricted motion in porcine HDL than in the human HDL2 and HDL3 subclasses. The differences in the spin label motion could not be accounted for on the basis of the differences in the chemical composition of the lipoproteins examined. They are, however, most probably due to the specific properties of the interactions between lipids and proteins that differ among the lipoproteins.

Animals↗
PubMed2001

Conformation of the gamma subunit at the gamma-epsilon-c interface in the complete Escherichia coli F(1)-ATPase complex by site-directed spin labeling.

Structure-function relationships of the gamma-epsilon-c subunit interface of F(O)F(1) ATP synthase, a region of subunit interactions important in coupling between catalysis and transport, were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The EPR line widths and collision accessibilities of 18 spin-labeled, unique cysteine F(1) mutants from gammaLeu198 to gammaLeu215 indicate an alternating pattern in the mobility and accessibility parameters for positions gamma201-209, which is reminiscent of a beta-strand. Labels at positions gamma204 and gamma210 show tertiary contact upon F(1) binding to F(O) and gammaD210C has reduced coupling efficiency. gammaE208C could not be spin labeled, but the uncoupling effects of gammaE208K are suppressed by second-site mutations in the polar loop of subunit c [Ketchum, C. J. and Nakamoto, R. K. (1998) J. Biol. Chem. 273, 22292-22297]. The restricted mobility and accessibility of spin labels in the odd-numbered positions between gamma201 and gamma207 plus the 2-4-fold higher values in k(cat) for ATP hydrolysis of these same mutant F(1) indicate that the interactions of these residues with the epsilon subunit mediate its inhibitory activity. Disrupted interactions with epsilon subunit also cause reduced coupling efficiency. We propose a model for the gamma-epsilon-c interface of Escherichia coli F(O)F(1) ATP synthase in which side chains from the odd-numbered residues of the gammaLys201-gammaTyr207 beta-strand directly and functionally interact with the epsilon subunit, while the even-numbered, acidic residues gammaAsp204, gammaGlu208, and gammaAsp210 interact with the F(O) sector, probably with subunit c. gamma Subunit interactions with both subunits in this region are important for coupling efficiency.

Amino Acid Substitution↗
PubMed1991

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor. At normal ionic strength (micro = 186 mM), a decrease in ST-EPR intensity (increase in microsecond F-actin mobility) was clearly indicated upon photolysis of 1 mM caged ATP with a 50-ms, 351-nm laser pulse. This increase in mobility is due to the complete dissociation of Si from the actin filament. At low ionic strength (micro, = 36 mM), when about half the Si heads remain bound during ATP hydrolysis, no change in the actin mobility was detected, despite much faster motions of labeled S1 bound to actin. Therefore, we conclude that the active interaction of Si, actin,and ATP induces rotation of myosin heads relative to actin, but does not affect the microsecond rotational motion of actin itself, as detected at cys-374 of actin.

Actins↗