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PubMed1993

Effects of bile acids and taurine on the lipid fluidity of hepatic microsomes in normal and bile duct-ligated rats--a spin label study.

In cholestasis and obstructive jaundice, hepatic bile acids and taurine content increase significantly and bile acids change in compositions. For example, increases in cholic acid, beta-muricholic acid and taurine-conjugated bile acids occur in rats. In this study, to clarify the significance of changes in bile acid composition in obstructive jaundice, the effects of various bile acids and taurine on the fluidity of the hepatic microsomal membrane were studied by the electron spin resonance spin-label method in normal and bile duct-ligated (for 10 days) rats. The flow properties (order parameter) of the shallow and deep layers of the membrane were determined with 5- and 12-doxyl stearic acid, respectively. Bile acids mainly affected the fluidity of the shallow membrane layer. The fluidity of the hepatic microsomal membrane was decreased in bile duct-ligated rats. Conjugated cholic acid (taurine-conjugated cholic acid, glycine-conjugated cholic acid and non-conjugated beta-muricholic acid exerted effects that increased fluidity in comparison with non-conjugated cholic acid and chenodeoxycholic acid, a precursor of beta-muricholic acid. Taurine had no clear effect on fluidity. In conclusion, cholic acid conjugation and beta-muricholic acid formation may be mechanisms which protect against the decrease in lipid fluidity of hepatic biomembranes in cholestasis or obstructive jaundice. Taurine may also affect fluidity through the formation of taurine-conjugated bile acids.

Animals↗
PubMed1985

Chemical modification of methionine residues of the phosphatidylcholine transfer protein from bovine liver. A spin-label study.

The role of methionine residues in the interaction of the phosphatidylcholine transfer protein from bovine liver with phospholipid vesicles was investigated by specific modification of these residues with iodoacetamide. The modified protein was digested with cyanogen bromide in order to determine which methionine residues had become resistant to this cleavage. Automated Edman degradation on the digest indicated that after 72 h of reaction, Met-1 was modified for 80%, Met-73 for 50%, Met-109 for 20%, whilst Met-173 and Met-203 were found to be unmodified. This distinct modification did not result in any loss of phosphatidylcholine transfer activity. The interaction of the phosphatidylcholine transfer protein with phospholipid vesicles was investigated by making use of electron spin resonance spectroscopy. The interaction of unmodified protein with vesicles composed of phosphatidylcholine/phosphatidic acid/spin-labeled phosphatidylethanolamine (79:16:5, mol%) or composed of phosphatidylserine/spin-labeled phosphatidylethanolamine (95:5, mol%), gave an increase of about 50% in the rotation correlation time. A similar increase was observed with the modified protein. This interaction was further investigated by labeling Met-1 and Met-73 in the transfer protein with iodoacetamidoproxyl spin-label. Spin-labeling did not inactivate the transfer protein. In addition, the electron spin resonance spectra of the spin-labeled protein were not affected upon addition of vesicles composed of phosphatidylcholine/phosphatidic acid (80:20, mol%). These experiments strongly suggest that Met-1 and Met-73 are not part of the site that interacts with the membrane.

Androgen-Binding Protein↗
PubMed1999

Mechanochemical coupling in spin-labeled, active, isometric muscle.

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force? Pate and Cooke (1989. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.

Animals↗
PubMed2006

Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA.

We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca(2+)], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation at Ser16 or by micromolar [Ca(2+)]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca(2+)] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca(2+)].

Amino Acid Sequence↗
PubMed1974

Distribution and fate of synthetic lipid vesicles in the mouse: a combined radionuclide and spin label study.

Single compartmental spherules of various lipid constituents (vesicles), enclosing (99m)TcO(4) (-) as a radioactive marker, were injected intravenously into C(3)H mice, and the distribution of radioactivity was studied. About 25% of the administered radioactivity was present in the liver 5 min and 30 min after the injection of vesicles composed of phosphatidylcholine and gangliosides, which were sonicated for 5 min (standard preparation). About 10-20% of the radioactivity remained in the circulation. By use of a nonradioactive spin label (tempocholine) enclosed within vesicles, intact vesicles were demonstrated in the circulation for 46 min after intravenous injection. The distribution of radioactivity from (99m)TcO(4) (-) inside vesicles is very different from that of free (99m)TcO(4) (-) or of (99m)Tc sulfur colloid. Increase in the length of sonication or incorporation of cholesterol into the wall of the vesicles enhanced hepatic levels and reduced blood levels of radioactivity. These same manipulations also slowed the rate of transfer of (99m)TcO(4) (-) out of vesicles in dialysis experiments in vitro. Addition of phosphatidic acid, phosphatidylethanolamine, or phosphatidylserine to the standard constituents did not greatly alter the distribution of radioactivity in vivo but did increase the number and type of active coupling sites on the outside of the vesicle. The results indicate that vesicles might be valuable as carriers of diagnostic or therapeutic agents.

Animals↗
PubMed2005

Myocardial blood flow mapping in mice using high-resolution spin labeling magnetic resonance imaging: influence of ketamine/xylazine and isoflurane anesthesia.

Genetically modified mouse models of many human diseases reflecting cardiovascular alterations are currently available. To date, little information on absolute myocardial perfusion in mice is found in the literature. High-resolution quantitative myocardial blood flow maps (in-plane resolution 156 x 312 mum(2), slice thickness 1.5 mm) have been obtained noninvasively within 25 min at 4.7 T in 30 freely breathing C57/Bl6J mice using electrocardiogram- and respiration-gated spin labeling magnetic resonance imaging (MRI). Regional myocardial blood flow measurements were carried out, and the effects of isoflurane at two different concentrations and ketamine/xylazine anesthesia were assessed. The mean blood flow value in the left ventricular myocardium was 6.0 +/- 1.9 mL g(-1) min(-1) under ketamine/xylazine and 6.9 +/- 1.7 mL g(-1) min(-1) (group average +/- SD) under isoflurane (1.25%). Under the influence of higher isoflurane concentration (2.00%), myocardial blood flow increased dramatically to 16.9 +/- 1.8 mL g(-1)min(-1) with no significant change in heart rate. This work illustrates the feasibility of noninvasive quantitative myocardial perfusion mapping in mice using MRI. The study of the influence of anesthesia shows that myocardial blood flow is highly sensitive to isoflurane concentration. The method employed offers a noninvasive approach to longitudinal studies of murine models of cardiac disease.

Adrenergic alpha-Agonists↗
PubMed2006

Spin labeling analysis of amyloids and other protein aggregates.

Because of the enormous size of amyloid fibrils and their low tendency to form crystal lattices, it has been difficult to obtain high-resolution structural information on these aggregates. Magnetic resonance methods, such as solid-state nuclear magnetic resonance spectroscopy and electron paramagnetic resonance (EPR) spectroscopy, are promising new technologies by which to obtain molecular models. This chapter will focus on the application of EPR spectroscopy to amyloids and other protein aggregates. Site-directed spin labeling (SDSL), in combination with EPR spectroscopy, has been successfully used to study protein structure and the dynamics of soluble as well as membrane proteins. Recent studies indicate that this strategy is also well suited for studying amyloid fibrils. For example, an important outcome of the SDSL studies performed in our laboratory is that fibrils of amyloid beta, islet amyloid polypeptide, alpha-synuclein, and tau have their beta-strands aligned in an in-register, parallel fashion. Future studies promise to yield molecular information about fibril topography and protofilament arrangement and can be extended to include oligomeric structures.

Amyloid↗
PubMed2006

Inverse correlation between cerebral blood flow measured by continuous arterial spin-labeling (CASL) MRI and neurocognitive function in children with sickle cell anemia (SCA).

Overt stroke, clinically "silent" cerebral infarct, and neurocognitive impairment are frequent complications of sickle cell anemia (SCA). Current imaging techniques have limited sensitivity and specificity to identify children at risk for neurocognitive impairment. We prospectively evaluated 24 children with SCA with a neurologic exam, complete blood count, transcranial Doppler ultrasound (TCD), measurement of intelligence quotient (IQ), and magnetic resonance imaging (MRI) with measurement of cerebral blood flow (CBF) using continuous arterial spin-labeling (CASL) MRI. Average CBF to gray matter was 112 +/- 36 mL/100 g/min. We identified a strong inverse relationship between performance IQ and CBF (-1.5 points per 10 mL/100 g/min increase in CBF, P = .013). Elevated steady-state white blood cell count (> or = 14 x 10(9)/L [14,000/microL]) was associated with lower full scale IQ (86 +/- 9 vs 99 +/- 10, P = .005). CASL MRI may identify children with neurocognitive impairment, before damage is evident by structural MRI or TCD.

Anemia, Sickle Cell↗
PubMed1993

Fluidity of chicken ventricular plasma membranes during development in-ovo and after birth: spin labelling and fluorescence studies.

The fluidity of isolated chicken ventricular plasma membranes was investigated by ESR spectroscopy, using three doxylstearic spin labels (5-SASL, 10-SASL and 16-SASL), and by fluorescence anisotropy measurements, using DPH probe. The analysis of 2T1 splitting constant and rotational correlation frequency have shown differences, during in-ovo development, characterized by a rigidification phase with a maximum at 19 days of development, just before hatching, followed by a fluidification phase after birth. These phenomena were also improved by DPH fluorescence and dynamical analysis of the hydrophobic 16-SASL probe.

Animals↗
PubMed2003

Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy.

Lipid rafts in the plasma membrane, domains rich in cholesterol and sphingolipids, have been implicated in a number of important membrane functions. Detergent insolubility has been used to define membrane "rafts" biochemically. However, such an approach does not directly contribute to the understanding of the size and the lifetime of rafts, dynamics of the raft-constituent molecules, and the function of rafts in the membrane in situ. To address these issues, we have developed pulse EPR spin labeling and single molecule tracking optical techniques for studies of rafts in both artificial and cell membranes. In this review, we summarize our results and perspectives obtained by using these methods. We emphasize the importance of clearly distinguishing small/unstable rafts (lifetime shorter than a millisecond) in unstimulated cells and stabilized rafts induced by liganded and oligomerized (GPI-anchored) receptor molecules (core receptor rafts, lifetime over a few minutes). We propose that these stabilized rafts further induce temporal, greater rafts (signaling rafts, lifetime on the order of a second) for signaling by coalescing other small/unstable rafts, including those in the inner leaflet of the membrane, each containing perhaps one molecule of the downstream effector molecules. At variance with the general view, we emphasize the importance of cholesterol segregation from the liquid-crystalline unsaturated bulk-phase membrane for formation of the rafts, rather than the affinity of cholesterol and saturated alkyl chains. In the binary mixture of cholesterol and an unsaturated phospholipid, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase, forming cholesterol-enriched domains or clustered cholesterol domains, probably due to the lateral nonconformability between the rigid planar transfused ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at C9-C10. However, such cholesterol-rich domains are small, perhaps consisting of only several cholesterol molecules, and are short-lived, on the order of 1-100 ns. We speculate that these cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein and cholesterol-rich domain was evaluated to be on the order of 100 micro, again showing the short lifetime of rafts in an unstimulated state. Finally, we propose a thermal Lego model for rafts as the basic building blocks for signaling pathways in the plasma membrane.

Animals↗
PubMed2004

The use of spin-labeled ligands as biophysical probes to report real-time endocytosis of G protein-coupled receptors in living cells.

Recycling and degradation of plasma membrane receptors and transporters are fundamental mechanisms for regulating cell signaling and metabolic processes. For many membrane proteins, endocytosis reduces the number of molecules available for transport or signal transduction, providing an attenuation response. Fluorescent reporters attached to either the receptor or ligand have been used to monitor the trafficking of internalization; however, these approaches provide poor resolution for the early endocytic response. Here, we describe the use of a spin-labeled ligand for a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor for measuring the kinetics of endocytosis in real time. Included are protocols for designing a nitroxide-labeled ligand and measuring receptor endocytosis in live cells using electron paramagnetic resonance (EPR) spectroscopy. Methods for the evaluation of the receptor binding and activation properties of modified ligands and the generation of a cell line stably expressing high receptor levels are also provided.

Animals↗
PubMed1980

Spin-label Studies of Membranes in Rye Protoplasts during Extracellular Freezing.

Protoplasts isolated from epicotyls of nonhardened winter rye seedlings were spin-labeled with the N-oxyl-4-4-dimethyloxazolidine derivatives of 5-ketostearic (5NS) and 16-ketostearic (16NS) acids. Spectra of the membrane-bound labels showed motional broadening with a rotational correlation time of 1.5 x 10(-8) second for 5NS and 1.5 x 10(-10) second for 16NS at 0 C. A procedure was developed to follow membrane changes in these protoplasts during extracellular freezing. With freezing, molecular motion of 5NS, but not of 16NS, spin probes was restricted. The increase in molecular order near the hydrated end of the membrane did not result from lowered temperatures inasmuch as no such change was observed in supercooled samples. These changes are probably due to dehydration of protoplast membranes during extracellular freezing. Similar results were obtained with multilayered egg yolk lecithin and are consistent with previous observations of changes in lecithin multibilayers during dehydration. Such alterations in membrane order might lead to irreversible membrane damage during extracellular freezing of plant cells.

Journal Article↗
PubMed2006

Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling.

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.

Bacterial Proteins↗
PubMed2000

Spin label EPR structural studies of the N-terminus of alpha-spectrin.

Spectrin, a vital component in human erythrocyte, is composed of alpha- and beta-subunits, which associate to form (alphabeta)2 tetramers. The tetramerization site is believed to involve the alpha-spectrin N-terminus and the beta-spectrin C-terminus. Abnormal interactions in this region may lead to blood disorders. It has been proposed that both termini consist of partial structural domains and that tetramerization involves the association of these partial domains. We have studied the N-terminal region of a model peptide for alpha-spectrin by making a series of double spin-labeled peptides and studying their dipolar interaction by electron paramagnetic resonance methods. Our results indicate that residues 21-42 of the N-terminus region exhibit an alpha-helical conformation, even in the absence of B-spectrin.

Biopolymers↗
PubMed2005

Noninvasive characterization of myocardial blood flow in diabetic, hypertensive, and diabetic-hypertensive rats using spin-labeling MRI.

OBJECTIVE: Microvascular alterations in the diabetic and hypertensive heart are likely to contribute to heart failure. In this work, myocardial blood flow and left ventricular function were measured in vivo in diabetic, hypertensive, and diabetic-hypertensive rats using MRI methods. METHODS: An 8-week-duration type 1 diabetes was induced by streptozotocin (STZ) in 8 Wistar-Kyoto (WKY) rats (STZ) and in 11 spontaneously hypertensive (SHR) rats (STZ-SHR). Fourteen WKY and 12 SHR served as control and hypertensive groups. Myocardial blood flow quantification was performed using an arterial spin-labeling MRI method. Left ventricular morphology and function were assessed during the same experiment using cine-MRI. RESULTS: Respective myocardial blood flow values for each group were 6.4 +/- 1.1 (WKY), 6.0 +/- 1.9 (STZ), 5.5 +/- 1.3 (SHR), and 4.3 +/- 0.9 mL. g(-1). min(-1) (STZ-SHR). Myocardial blood flow was significantly decreased in STZ-SHR rats compared with the other groups (p <.05, STZ-SHR vs. all groups). Cine-MRI showed morphological alterations in all pathological groups. No alteration of the ejection fraction was observed in the pathological groups. CONCLUSION: Myocardial blood flow is altered in vivo before any sign of heart failure when rats have type 1 diabetes and hypertension simultaneously. When only one of the pathologies occurs, MBF does not vary significantly.

Animals↗
PubMed2006

Simultaneous laser Doppler flowmetry and arterial spin labeling MRI for measurement of functional perfusion changes in the cortex.

This study compares laser Doppler flowmetry (LDF) and arterial spin labeling (ASL) for the measurement of functional changes in cerebral blood flow (CBF). The two methods were applied concurrently in a paradigm of electrical whisker stimulation in the anaesthetised rat. Multi-channel LDF was used, with each channel corresponding to different fiber separation (and thus measurement depth). Continuous ASL was applied using separate imaging and labeling coils at 3 T. Careful experimental set up ensured that both techniques recorded from spatially concordant regions of the barrel cortex, where functional responses were maximal. Strong correlations were demonstrated between CBF changes measured by each LDF channel and ASL in terms of maximum response magnitude and response time-course within a 6-s-long temporal resolution imposed by ASL. Quantitatively, the measurements of the most superficial LDF channels agreed strongly with those of ASL, whereas the deeper LDF channels underestimated consistently the ASL measurement. It was thus confirmed that LDF quantifies CBF changes consistently at a superficial level, and for this case the two methods provided concordant measures of functional CBF changes, despite their essentially different physical principles and spatiotemporal characteristics.

Animals↗
PubMedDate not supplied

Cerebral perfusion MRI with arterial spin labeling technique at 0.5 Tesla.

PURPOSE: Our aim was to evaluate the feasibility of cerebral perfusion MRI using an arterial spin labeling technique at 0.5 T. METHOD: We performed perfusion imaging with a flow-sensitive alternating inversion recovery (FAIR) sequence in a total of 37 patients with cerebral infarction. RESULTS: FAIR perfusion images demonstrated areas of pathological perfusion corresponding (13 patients) or not corresponding (15 patients) to the infarcted area on MR images. Among 19 patients in whom comparison between FAIR perfusion imaging and regional cerebral blood flow single photon emission CT was available, the two studies correlated well in 15 patients. CONCLUSION: Our results indicate that the FAIR technique allows reliable cerebral perfusion imaging at 0.5 T.

Adolescent↗
PubMed2004

Synthesis and enzyme-catalyzed hydrolysis of a radical-masked glycosylated spin-label reagent.

N1-Acetoxy-2,2,6,6-tetramethylpiperidin-4-yl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosides (3-alpha, beta) and N1-acetoxy-2,2,5,5-tetramethylpyrrolin-3-oyl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosylamines (9-alpha, beta) were synthesized in good yield by Schmidt's glycosylation method. Their subsequent O-debenzylation was proceeded successfully to give the desired products 1-alpha, and 1-beta in good yield, and 2-alpha in a low yield, without 2-beta by only short-timed hydrogenolysis in the presence of palladium-on-carbon (Pd-C) in a CHCl3-MeOH solvent system that included concentrated HCl. Upon enzyme-catalyzed hydrolysis, only 2-alpha was hydrolyzed by the esterase, while both of 1-alpha and 1-beta were not hydrolyzed by any other enzyme such as lipase. These 2-alpha can likely be used as a new water-soluble radical-masked glycosylated spin-label reagent.

Catalysis↗