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PubMed1982

Vitamin D-mediated intestinal calcium transport. Effects of essential fatty acid deficiency and spin label studies of enterocyte membrane lipid fluidity.

Vitamin D-3 and its metabolites regulate the transport of calcium across the intestinal epithelial cell via a mechanism which is as yet unknown. The purpose of this study was to evaluate the effect of an essential fatty acid deficiency on vitamin D-stimulated intestinal calcium transport as measured by both in vivo and in vitro techniques. We also describe in this report a procedure for the isolation of chick intestinal epithelial cell brush border and basal lateral membranes and an assessment of the effect of dietary vitamin D on the lipid fluidity of these membranes. An essential fatty acid deficiency in both vitamin D-replete and deficient chicks resulted in a decrease in intestinal mucosal levels of linoleic acid, with a compensatory increase in the levels of the short chain fatty acid, myristic acid, and the unsaturated fatty acids, palmitoleic and eicosatrienoic acids. An essential fatty acid deficiency did not affect the ability of vitamin D-deficient chicks to respond to vitamin D with a 2-fold increase in serum calcium and a 4-5-fold increase in intestinal calcium transport, measured in vivo. However, an essential fatty acid deficiency resulted in an inability of vitamin D to increase calcium efflux in vitamin D-deficient chick ileum as measured under in vitro conditions. Dietary vitamin D resulted in no detectable change in the protein composition in either the brush border or basal lateral membranes as evidenced by SDS-polyacrylamide electrophoresis. In addition, vitamin D did not alter the levels of brush border membrane cholesterol or lipid phosphorus (0.27 +/- 0.03 and 0.19 +/- 0.01 mumol/mol protein, respectively). Brush border and basal lateral membranes were labeled with the 5-nitroxide stearate spin probe I(12,3). The polarity of the environment of the probe in the brush border membranes is much greater than that of the basal lateral membranes. In addition, the lipid environment of the brush border membrane is much less fluid (S = 0.650) that that of the basal lateral membrane (S = 0.583). The data concerning membrane lipid fluidity is qualitatively similar to fluorescence polarization studies of rat intestinal epithelial cell membranes and confirms the concept that a given cell may contain plasma membrane regions having discrete lipid structures/fluidities. Dietary vitamin D had no detectable effect on the lipid fluidity or polarity in either the brush border or basal lateral membranes. The results do not support a role for an alteration in essential fatty acid composition or gross changes in the lipid fluidity of the brush border or basal lateral membranes as mechanisms by which vitamin D regulates intestinal calcium transport.

Animals↗
PubMed1981

Spin-label detection of hemoglobin-membrane interaction at physiological pH.

The interaction between hemoglobin and the cytoplasmic surface of human erythrocyte membranes at physiological pH was studied by monitoring the electron paramagnetic resonance (EPR) signal of spin-labeled membrane ghosts in hemoglobin solutions of various concentrations. The EPR spectra indicate the existence of a significant hemoglobin-membrane interaction which exhibits a substantial hemoglobin concentration dependence over the concentration range 0-12 mg/mL. An equilibrium binding model yields a hemoglobin-membrane dissociation constant, Kd, on the order of 10(-4) M, at and above physiological pH; the interaction is classified as very low-affinity binding. The interaction increases significantly when the pH is decreased. Half-saturation of the binding sites occurs at a ratio of about 10(8) hemoglobins per cell.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Träuble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.

Animals↗
PubMed1982

Reactivity of sarcoplasmic reticulum adenosinetriphosphatase with iodoacetamide spin-label: evidence for two conformational states of the substrate binding sites.

The labeling kinetics of sarcoplasmic reticulum ATPase with the iodoacetamide spin probe N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide were followed under conditions designed to selectively label all reactive groups. Approximately 1 mol of spin-label reacted per one 100 000-dalton ATPase chain, indicating only one residue on the enzyme had been labeled. One uniform rate of labeling was observed in the presence of Ca2+. When substrate was then added, approximately one-half of the residues showed a 10-fold increase in labeling rate while the remaining residues reacted at the initial, slower rate. Sequential labeling experiments further established that the two labeling rates correspond to the coexistence of two conformational state of the enzyme. Both Ca2+ and substrate are required to obtain an equal distribution between states, and the effect is completely reversed when substrate is removed. The iodoacetamide spin probe is known to be highly sensitive to the conformation of the ATPase binding pocket, and the residue labeled here is the one which generates broadening in the electron paramagnetic resonance spectrum on substrate binding. Due to the unique selectively of the labeling reaction, it is suggested that when both substrate and Ca2+ are bound to the enzyme, conditions which are precursory to enzyme phosphorylation, two specific conformations of the binding pocket exist in approximately at 50:50 ratio.

Adenosine Triphosphatases↗
PubMed1977

An electron spin resonance study of cholestane spin label in aqueous mixtures of biliary lipids.

The effect of cholesterol on the fluidity of the phospholipid matrix in mixed micelles derived from bile salts and lecithin has been determined by the paramagnetic probe technique. It was found that correlation times for the cholestane spin label were discontinuous functions of cholesterol content and that these discontinuities correlate with the equilibrium solubility limit for cholesterol in this quaternary system. The origin of these discontinuities is attributed to the existence of another aggregate in addition to the discshaped mixed micelle in lipid solutions supersaturated with cholesterol.

Bile↗
PubMedDate not supplied

[Study of erythrocyte dehydration using spin labels].

Possibility of studying erythrocyte dehydration by ESR-spin probe is substantiated. Dehydration of erythrocytes in relation to osmolarity of sodium chloride solutions is investigated. The results are shown to agree with the data obtained by radioisotope method.

Body Fluids↗
PubMed1990

Influence of polar residue deletions on lipid-protein interactions with the myelin proteolipid protein. Spin-label ESR studies with DM-20/lipid recombinants.

The lipid specificities of two related integral membrane proteins of central nervous system myelin, the proteolipid (PLP) and DM-20 proteins, which differ only by the deletion of a polar stretch of 35 contiguous amino acid residues, were studied with spin-labeled lipids after reconstitution into dimyristoyl-phosphatidylcholine. The selectivity in populating lipid association sites at the protein interface and in modulating the lipid exchange between protein and bulk lipid sites was quantitated by the relative association constants and the off-rate constants for exchange, respectively, for both proteins. The sequence deleted in DM-20 (residues 116-150 of PLP) is found to play a major role in determining the lipid selectivity for the parent PLP protein.

Animals↗
PubMed1982

A spin label study of the lipid boundary layer of mitochondrial NADH-ubiquinone oxidoreductase.

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I) is a lipoprotein enzyme containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin. Enzyme preparations containing endogenous cardiolipin and a range of either soyabean PC or dimyristoylphosphatidylcholine (DMPC) concentrations have been made. Using a spin-labelled fatty acid, two probe environments differing in mobility have been shown to be present. The fatty acid probe has a relative binding constant (or partition coefficient between lipid and protein) of unity. The boundary layer or lipid annulus reported by the probe has a value of approx. 300 lipid molecules per molecule of enzyme FMN in preparations containing soyabean PC, or DMPC above the phase transition temperature of the latter. In soyabean PC-replaced enzyme the apparent size of the boundary layer is independent of temperature between 30 degrees C and 14 degrees C but shows a modest increase to about 400 lipid molecules per molecule of FMN between 14 degrees C and 2 degrees C. Complex I replaced with high concentrations of DMPC gives non-linear Arrhenius plots of NADH-ubiquinone oxidoreductase activity. The results of the ESR experiments show that both boundary layer and bulk lipid must be motionally restricted for this to occur. Thus, the change in activity is probably not caused by an effect exerted directly on the catalytic activity of the enzyme but is more likely due to restriction of free diffusion of ubiquinone to its site of reduction.

Animals↗
PubMed1969

Orientation and motion of amphiphilic spin labels in membranes.

The paramagnetic resonance spectra of N-oxyl-4','-dimethyloxazolidine derivatives of 12-ketostearic acid, 5-ketostearic acid, 5-ketotricosanoic acid, and 5alpha-androstan-3-one-17beta-ol have been observed in sonicated phospholipid dispersions, the walking-leg nerve fibers of Homarus americanus, and in erythrocytes oriented by hydrodynamic shear. The preferred orientation of these spin labels in the nerve and erythrocyte membranes is one in which the long amphiphilic axis is perpendicular to the membrane surface. The resonance spectra indicate that these labels in the nerve fiber are bound to a lipid bilayer very similar to that found in the sonicated phospholipid dispersion, whereas any lipid bilayer in the erythrocyte must be much more rigid.

Animals↗
PubMed1984

Effect of tumour angiogenesis factors on the membrane of capillary and aortic endothelial cells: an e.s.r. spin-label study.

Two distinct factors have been separated from an angiogenic extract of a rat Walker 256 carcinoma, one inducing proliferation and the other migration of capillary endothelial cells in vitro, but having no detectable effect on aortic endothelial cells. The influence of these factors on the order of plasma membranes of these cells was examined by electron spin resonance, using the lipophilic spin label 5-doxyl stearic acid. No detectable effect was observed on treating whole cells or isolated membranes with proliferation factor. In contrast, exposure of capillary endothelial cell membranes to migration factor caused a reduction of membrane order, particularly at temperatures above 30 degrees C. The migration factor had no detectable effect on membrane order of aortic endothelial cells.

Angiogenesis Inducing Agents↗
PubMed1972

Mechanism of cooperative oxygen binding to hemoglobin (spin-labeled triphosphate-concerted transition model-hemoglobin chesapeake).

Evidence is presented that a generalized concerted transition model provides a quantitative understanding of (a) the molecular species that are present in solutions of partially liganded hemoglobin and (b) the macromolecular mechanism of cooperativity. Model parameters for hemoglobin A and for hemoglobin Chesapeake were determined from studies of the binding of spin-label triphosphates to ligand-free and partially liganded hemoglobin solutions, and to the hybrids alpha(2) (+CN)beta(2) and alpha(2)beta(2) (+CN). This model is the same as that proposed originally by Monod, Wyman, and Changeux [J. Mol. Biol. (1965) 12, 88] for hemoglobin, except that the alpha-subunits are treated as nonequivalent to the beta-subunits.

Electron Spin Resonance Spectroscopy↗
PubMed2006

Pediatric perfusion MR imaging using arterial spin labeling.

Cerebral blood flow (CBF, cerebral perfusion) mirrors cerebral metabolic demand and neuronal function, and therefore, is a vital parameter in the evaluation of pediatric brain injury and recovery. Until recently, measurement of CBF involved intravenous bolus injection of contrast agents or nuclear medicine methods that were technically difficult or ethically problematic in pediatrics. The development of arterial spin label (ASL) perfusion MR imaging as a noninvasive method for measuring CBF allows for the increased ability to measure this vital physiologic parameter in any age group. This article presents the technical aspects of performing ASL perfusion MR imaging in pediatrics, and discusses its current use in clinical studies and its potential for influencing important management strategies in specific disease entities.

Anemia, Sickle Cell↗
PubMed2004

Assessment of irradiated brain metastases by means of arterial spin-labeling and dynamic susceptibility-weighted contrast-enhanced perfusion MRI: initial results.

RATIONALE AND OBJECTIVES: To assess if preradiation and early follow-up measurements of relative regional cerebral blood flow (rrCBF) can predict treatment outcome in patients with cerebral metastases and to evaluate rrCBF changes in tumor and normal tissue after stereotactic radiosurgery using arterial spin-labeling (ASL) and first-pass dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion MRI. METHODS: In 25 patients with a total of 28 brain metastases, DSC MRI and ASL perfusion MRI using the Q2TIPS sequence were performed with a 1.5-T unit. Measurements were performed prior to and at 6 weeks, 12 weeks, and 24 weeks after stereotactic radiosurgery. Follow-up examinations were completely available in 25 patients for Q2TIPS and 17 patients with 18 metastases for DSC MRI. The rrCBF of the metastases and the normal brain tissue was determined by a region-of-interest analysis. rrCBF values were correlated with the treatment outcome that was classified according to tumor volume changes at 6 months. RESULTS: The alteration of the rrCBF at the 6-week follow-up was highly predictive for treatment outcome. A decrease of the rrCBF value predicted tumor response correctly in all metastases for Q2TIPS and in 13 of 16 metastases for DSC MRI. The pretherapeutic rrCBF was not able to predict treatment outcome. The rrCBF values in normal brain tissue affected by radiation doses less than 0.5 Gy remained unchanged after therapy. CONCLUSION: These preliminary results suggest that ASL and DSC MRI techniques determining rrCBF changes in brain metastases after stereotactic radiosurgery allow the prediction of treatment outcome.

Adult↗
PubMed1999

Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane

The physical characteristics of rubber particles from the four rubber (cis-1,4-polyisoprene) producing species Euphorbia lactiflua Phil., Ficus elastica Roxb., Hevea brasiliensis Mull. Arg., and Parthenium argentatum Gray, were investigated using transmission electron microscopy (TEM) and electron-paramagnetic-resonance (EPR) spin labeling spectroscopy. Transmission electron microscopy showed the rubber particles to be composed of a spherical, homogeneous, core of rubber enclosed by a contiguous, electron-dense, single-track surface layer. The biochemical composition of the surface layer and its single-track TEM suggested that a monolayer biomembrane was the surface structure most compatible with the hydrophobic rubber core. The EPR spectra for a series of positional isomers of doxyl stearic acid, used to label the surface layer of the rubber particles, exhibited flexibility gradients and evidence for lipid-protein interactions for all four rubber particle types that is consistent with a biomembrane-like surface. The EPR spectra confirmed that the surface biomembrane is a monolayer. Thus, rubber particles appear similar to oil bodies in their basic architecture. The EPR spectra also provided information on protein location and degree of biomembrane penetration that correlated with the known properties of the rubber-particle-bound proteins. The monolayer biomembrane serves as an interface between the hydrophobic rubber interior and the aqueous cytosol and prevents aggregation of the particles. An unexpected observation for the probes in pure polyisoprene was evidence of an intrinsic flexibility gradient associated with the stearic acid molecule itself.

Journal Article↗
PubMedDate not supplied

Speeding up a genetic algorithm for EPR-based spin label characterization of biosystem complexity.

Complexity of biological systems is one of the toughest problems for any experimental technique. Complex biochemical composition and a variety of biophysical interactions governing the evolution of a state of a biological system imply that the experimental response of the system would be superimposed of many different responses. To obtain a reliable characterization of such a system based on spin-label Electron Paramagnetic Resonance (EPR) spectroscopy, multiple Hybrid Evolutionary Optimization (HEO) combined with spectral simulation can be applied. Implemented as the GHOST algorithm this approach is capable of handling the huge solution space and provides an insight into the "quasicontinuous" distribution of parameters that describe the biophysical properties of an experimental system. However, the analysis procedure requires several hundreds of runs of the evolutionary optimization routine making this algorithm extremely computationally demanding. As only the best parameter sets from each run are assumed to contribute into the final solution, this algorithm appears far from being optimized. The goal of this study is to modify the optimization routine in a way that 20-40 runs would be enough to obtain qualitatively the same characterization. However, to keep the solution diversity throughout the HEO run, fitness sharing and newly developed shaking mechanisms are applied and tested on various test EPR spectra. In addition, other evolutionary optimization parameters such as population size and probability of genetic operators were also varied to tune the algorithm. According to the testing examples a speed-up factor of 5-7 was achieved.

Algorithms↗
PubMed1983

A spin-label and hydrogen-deuterium exchange reaction kinetics study of protein-lipid interactions in lipid-replaced Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.

The Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20 degrees C and 30 degrees C, respectively, in accord with the results of previous investigators. Measurement by the spin-labeling technique of the fluidity of the bulk lipid revealed the gel-to-liquid crystalline phase transition at 29 degrees C and 39 degrees C, respectively, while the fluidity of the boundary lipid in both samples was found to be low throughout the temperature range studied. The rotational mobility of the Ca2+-ATPase protein in both samples, measured by saturation transfer electron spin resonance, was also very low throughout the temperature range studied and its temperature-dependence did not show any break or jump corresponding to the phase transition of the bulk lipid. On the other hand, the structural fluctuation of the Ca2+-ATPase protein in dimyristoylphosphatidylcholine-recombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27 degrees C, apparently in accordance with the phase transition of the bulk lipid. Results obtained in this study suggested that the Ca2+-ATPase protein molecules are in an aggregated state in these reconstituted membranes and that the Ca2+-ATPase activity is neither directly correlated to the fluidity of the boundary lipid nor to the rotational mobility of the Ca2+-ATPase, contrary to the suggestions of previous investigators (Hesketh et al. (1976) Biochemistry 15, 4145-4151; Hidalgo et al. (1978) J. Biol. Chem. 253, 6879-6887).

Animals↗
PubMed2006

L-carnitine inhibits apoptotic DNA fragmentation induced by a new spin-labeled derivative of podophyllotoxin via caspase-3 in Raji cells.

L-carnitine (beta-hydroxy-trimethylaminobutyric acid) plays an essential metabolic role that consists of transferring the long chain fatty acids through the mitochondrial barrier, thus allowing their energy-yielding oxidation. GP7 (4-[4''-(2'', 2'', 6'', 6''-tetramethyl-l''-piperidinyloxy) amino] -4'-demethyl-epipodophyllotoxin) is a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university. In this study, we examined the activity of L-carnitine in GP7-induced apoptosis in Burkitt's lymphoma cell line, Raji. GP7 induced time- and dose-dependent apoptotic DNA fragmentation accompanied by caspase-3 activation in Raji cells, and the kinetics of caspase-3 activation induced by GP7 was well correlated with that of apoptotic DNA fragmentation. L-carnitine treatment prevented GP7-induced caspase-3 activation, suppressed caspase-3 cleavage and abrogated GP7-induced apoptotic DNA fragmentation in Raji cells. Our findings suggest that L-carnitine is a potent anti-apoptotic agent to human lymphoma cells and may exert its anti-apoptotic effect via inhibition of caspase-3 activity in GP7-treated Raji cells.

Apoptosis↗