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Biomedical subjects

D Marsh

Publications and source records attributed to D Marsh.

At least 19 recordsLinked to original sources
PubMed2006

Multifrequency simulations of the EPR spectra of lipid spin labels in membranes.

Simulations are performed of 34- and 9-GHz EPR spectra, together with 94-GHz EPR spectra, from phospholipid probes spin-labelled at the C4-C14 positions of the sn-2 chain, in liquid-ordered and gel-phase membranes of dimyristoyl phosphatidylcholine with high and low cholesterol contents. The multifrequency simulation strategy involves: (i) obtaining partially averaged spin-Hamiltonian tensors from fast-motional simulations of the 94-GHz spectra; (ii) performing slow-motional simulations of the 34- and 9-GHz spectra by using these pre-averaged tensors with the stochastic Liouville formalism; (iii) constructing, by simulation, slow-motional calibrations for the differences, DeltaA(zz)(qx) and Deltag(zz)(qx), in effective A(zz)-hyperfine splittings and g(zz)-values between 34- (or 94-GHz) and 9-GHz spectra; (iv) using such calibrations for DeltaA(zz)(qx) and Deltag(zz)(qx) and dynamic parameters from stage (ii) as a guide to adjust the extent of pre-averaging of the spin-Hamiltonian tensors; and (v) repeating the 34- and 9-GHz simulations of stage (ii). By using this scheme it is possible to obtain consistent values of the rotational diffusion coefficients, D(R perpendicular) and D(R//), and the long-axis order parameter, S(zz), that characterize the slow axial motion of the lipid chains, from spectra at both 34 and 9GHz. Inclusion of spectra at 34GHz greatly improves precision in determining the D(R//) element of the slow diffusion tensor in these systems.

Computer Simulation↗
PubMed2005

Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance.

Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.

Electron Spin Resonance Spectroscopy↗
PubMed2005

Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values.

The use of 90 degrees-out-of-phase first-harmonic absorption (V1'-) EPR to resolve the spectra from nitroxide spin labels with differing T1-relaxation times is described. Non-linear V1'-EPR spectra recorded under moderate saturation have sharper lines compared with the in-phase V1-EPR spectra, and amplitudes that preferentially enhance components with longer T1-relaxation. Discrimination between V1'-spectral components can be increased further by means of selective paramagnetic relaxation enhancement agents. Examples are given of biophysical applications to double labelling in single-component membranes and phase separation in two-component membranes, to lipid-peptide complexes, and to binding of spin-labelled reagents. It is concluded that optimal resolution in V1'-EPR spectroscopy is obtained at relatively low Zeeman modulation frequencies (20-30 kHz) and moderate saturation (H1 approximately 0.2-0.3 G).

Absorption↗
PubMed2004

Membrane penetration of nitric oxide and its donor S-nitroso-N-acetylpenicillamine: a spin-label electron paramagnetic resonance spectroscopic study.

S-nitroso-N-acetylpenicillamine (SNAP) is a pharmacological agent with diverse biological effects that are mainly attributable to its favorable characteristics as a nitric oxide (NO)-evolving agent. It is found that SNAP incorporates readily into dimyristoyl phosphatidylcholine (DMPC) bilayer membranes; and an approximate penetration profile was obtained from the depth dependence of the perturbation that it exerts on spin-labeled lipid chains. The profile of SNAP locates it deep in the hydrophobic core of both fluid- and gel-phase membranes. The spin relaxation enhancement of spin-labeled phospholipids with nitroxide group located at different depths in DMPC membranes was determined for nitric oxide (NO) and molecular oxygen (O(2)), at close to atomic spatial resolution. The relaxation enhancement, which is proportional to the corresponding vertical membrane profile of the concentration-diffusion product, was measured in the gel and fluid phases of the lipid bilayer. No significant membrane penetration was observed in the gel phase for the two water-dissolved gases. In the fluid phase, the transmembrane profiles of NO and O(2) are similar and could be well described by a sigmoidal function with a maximum in the center of the bilayer, but that of NO is less steep and is shifted toward the center of the membrane, relative to that of O(2). These differences can be attributed mainly to the difference in hydrophobicity between the two gases and the presence of the donor in the NO experiments. The biological implications of the above results are discussed.

Cell Membrane↗
PubMed2003

Miscibility and phase behaviour of binary mixtures of N-palmitoylethanolamine and dipalmitoylphosphatidylcholine.

The content of N-acylethanolamines (NAEs) increases dramatically in cell membranes when the parent organism is subjected to injury or stress. This increase has been attributed to stress-combating mechanisms of the organism. In this study, a binary phase diagram of hydrated mixtures of N-palmitoylethanolamine (NP-E)--an endogenous ligand for the peripheral cannabinoid receptor (CB-2)--with dipalmitoylphosphatidylcholine (DPPC) is established by high-sensitivity differential scanning calorimetry (DSC). The structures of the phases involved were determined by using 31P-NMR spectroscopy and low-angle X-ray scattering. DSC studies show that NP-E and DPPC mix well in the composition range DPPC/NP-E=100:0 to 40:60 (mol/mol). At higher contents of NP-E, phase separation is indicated by the presence of additional transitions in the thermograms. Characterization of the structures formed by the mixtures with 31P-NMR shows that, up to 80 mol% NP-E, DPPC remains in the lamellar phase. The low-angle X-ray diffraction data are also consistent with a lamellar gel-phase structure for DPPC/NP-E mixtures up to 60 mol% NP-E. Above 70 mol% NP-E, NP-E phase separates in the gel-phase region, while complete miscibility is observed in the fluid phase. These results provide a structural basis for understanding the membrane interactions of NAEs, which is necessary for understanding the mechanism of their putative stress-combating role in the parent organisms.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Lipid-protein interactions and effect of local anesthetics in acetylcholine receptor-rich membranes from Torpedo marmorata electric organ.

The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine >> tetracaine approximately procaine.

Anesthetics, Local↗
PubMed2003

Lipid interactions with transmembrane proteins.

Magnetic resonance results, principally from 2H-nuclear magnetic resonance, indicate that the mean lipid-chain ordering at the surface of transmembrane proteins is comparable to that in fluid lipid bilayers. Principally, it is the requirement for matching the hydrophobic lengths of lipid and protein that modulates the degree of chain ordering at the lipid-protein interface. The distribution of chain order parameters is, nonetheless, broader in the presence of integral proteins than in fluid lipid bi-layers. The chain configurations of the phospholipids that are resolved in crystals of integral membrane proteins display considerable conformational heterogeneity. Chain C-C dihedral angles are, however, not restricted to the energetically allowable trans and gauche rotamers.This indicates that the chains of a given lipid do not have a unique configuration in protein crystals.

Crystallization↗
PubMed2003

Predicting survival after treatment for fracture of the proximal femur and the effect of delays to surgery.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the prognosis after treatment for femoral neck fracture, to assess the impact of delay to surgery, and to devise a clinical prediction rule and score. METHODS: A prospective observational study was conducted in which 1780 patients treated surgically in two teaching hospitals between 1 November 1997 and 31 October 1999 were followed over 12 months. Logistic regression was used to distinguish the effects of predictor variables on survival. Using a probit transformation of the predicted posterior probabilities of death, a prognostic score was devised with scores constrained so that a nominal score of approximately 90 represented a 50:50 chance of survival over 12 months. RESULTS: Mortality was 30.1% in men and 19.5% in women. Increasing age, male gender, longer pre-operative delay, a higher American Society of Anesthesiology score, a lower Mental Test score, and a lower activities of daily living (Barthel) score were associated with increased risks of death. Of those waiting between 1 and 5 days for surgery, approximately 8 medium-risk and 17 high-risk patients (with prognostic scores of 90 and 120, respectively) would have to have their delay reduced to < 24 hours to yield one additional survivor. CONCLUSION: The application of prediction rules must be guided by ethical, social, and scientific concerns.

Activities of Daily Living↗
PubMed2003

Anisotropic motion effects in CW non-linear EPR spectra: relaxation enhancement of lipid spin labels.

Continuous-wave (CW) EPR measurements of enhancements in spin-lattice (T(1)-) relaxation rate find wide application for determining spin-label locations in biological systems. Often, especially in membranes, the spin-label rotational motion is anisotropic and subject to an orientational potential. We investigate here the effects of anisotropic diffusion and ordering on non-linear CW-EPR methods for determining T(1) of nitroxyl spin labels. Spectral simulations are performed for progressive saturation of the conventional in-phase, first-harmonic EPR signal, and for the first-harmonic absorption EPR signals detected 90 degrees -out-of-phase with respect to the Zeeman field modulation. Motional models used are either rapid rotational diffusion, or strong-jump diffusion of unrestricted frequency, within a cone of fixed maximum amplitude. Calculations of the T(1)-sensitive parameters are made for both classes of CW-experiment by using motional parameters (i.e., order parameters and correlation times), intrinsic homogeneous and inhomogeneous linewidth parameters, and spin-Hamiltonian hyperfine- and g-tensors, that are established from simulation of the linear CW-EPR spectra. Experimental examples are given for spin-labelled lipids in membranes.

Anisotropy↗
PubMed2002

Effect of cholesterol on the interaction of seminal plasma protein, PDC-109 with phosphatidylcholine membranes.

Binding of PDC-109, the major protein of the bovine seminal plasma, to sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, a necessary event before capacitation can occur. The selectivity of PDC-109 for different spin-labelled phospholipids and sterol probes in dimyristoylphosphatidylcholine (DMPC) host matrix has been characterized earlier by EPR spectroscopy [Ramakrishnan, M., Anbazhagan, V., Pratap, T.V., Marsh, D. and Swamy, M.J. (2001) Biophys. J. 81, 2215-2225]. In this report the effect of cholesterol on the interaction of PDC-109 with DMPC membranes has been investigated by spin-label EPR spectroscopy. The results indicate that the presence of cholesterol leads to an increased association of different phospholipid as well as sterol probes, thus modulating the interaction of PDC-109 with phospholipid membranes.

Animals↗
PubMed2002

Biomechanical model to simulate tissue differentiation and bone regeneration: application to fracture healing.

Bone regeneration is a common biological process occurring, for example, during fracture healing or osseo-integration of prostheses. Computer simulation of bone regeneration is difficult to carry out because it is a complex sequence of cell-mediated processes regulated by mechanobiological stimuli. An algorithm to predict the time-course of intramembranous and endochondral ossification has been developed. The algorithm assumes that there are precursor cells in the undifferentiated tissue and that these cells differentiate into either fibroblasts (to form fibrous connective tissue), chondrocytes (to form cartilaginous tissue) or osteoblasts (to form bone), based on a combination of biophysical stimuli derived from strain in the collagenous matrix and flow of the interstitial fluid. Both these stimuli are known to deform the precursor cells, and the authors hypothesise that this causes activation of cell differentiation pathways. The observation that precursor cells take time to spread throughout the fracture callus has been included in the algorithm. The algorithm was tested in an investigation of the fracture healing of a long bone using an axi-symmetric finite element model. The spatio-temporal sequence of tissue phenotypes that appear in the course of fracture healing was successfully simulated. Furthermore, the origin of the precursor cells (either surrounding muscle, bone marrow or periosteum) was predicted to have a fundamental effect on the healing pattern and on the rate of reduction of the interfragmentary strain (IFS). The initial IFS = 0.15 drops to 0.01 within seven iterations if cells originated from the surrounding soft tissue, but took more than 50% longer if cells originated in the inner cambium layer of the periosteum, and four times longer if precursor cells originated from the bone marrow only.

Algorithms↗
PubMed2002

Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients.

BACKGROUND: The treatment of open fractures of the tibial shaft is often complicated by delayed union and nonunion. The objective of this study was to evaluate the safety and efficacy of the use of recombinant human bone morphogenetic protein-2 (rhBMP-2; dibotermin alfa) to accelerate healing of open tibial shaft fractures and to reduce the need for secondary intervention. METHODS: In a prospective, randomized, controlled, single-blind study, 450 patients with an open tibial fracture were randomized to receive either the standard of care (intramedullary nail fixation and routine soft-tissue management [the control group]), the standard of care and an implant containing 0.75 mg/mL of rhBMP-2 (total dose of 6 mg), or the standard of care and an implant containing 1.50 mg/mL of rhBMP-2 (total dose of 12 mg). The rhBMP-2 implant (rhBMP-2 applied to an absorbable collagen sponge) was placed over the fracture at the time of definitive wound closure. Randomization was stratified by the severity of the open wound. The primary outcome measure was the proportion of patients requiring secondary intervention because of delayed union or nonunion within twelve months postoperatively. RESULTS: Four hundred and twenty-one (94%) of the patients were available for the twelve-month follow-up. The 1.50-mg/mL rhBMP-2 group had a 44% reduction in the risk of failure (i.e., secondary intervention because of delayed union; relative risk = 0.56; 95% confidence interval = 0.40 to 0.78; pairwise p = 0.0005), significantly fewer invasive interventions (e.g., bone-grafting and nail exchange; p = 0.0264), and significantly faster fracture-healing (p = 0.0022) than did the control patients. Significantly more patients treated with 1.50 mg/mL of rhBMP-2 had healing of the fracture at the postoperative visits from ten weeks through twelve months (p = 0.0008). Compared with the control patients, those treated with 1.50 mg/mL of rhBMP-2 also had significantly fewer hardware failures (p = 0.0174), fewer infections (in association with Gustilo-Anderson type-III injuries; p = 0.0219), and faster wound-healing (83% compared with 65% had wound-healing at six weeks; p =0.0010). CONCLUSIONS: The rhBMP-2 implant was safe and, when 1.50 mg/mL was used, significantly superior to the standard of care in reducing the frequency of secondary interventions and the overall invasiveness of the procedures, accelerating fracture and wound-healing, and reducing the infection rate in patients with an open fracture of the tibia.

Adolescent↗
PubMed2001

Specific surface association of avidin with N-biotinylphosphatidylethanolamine membrane assemblies: effect on lipid phase behavior and acyl-chain dynamics.

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.

Avidin↗
PubMed2001

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes.

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.

Avidin↗
PubMed2001

Polarity and permeation profiles in lipid membranes.

The isotropic (14)N-hyperfine coupling constant, a(o)(N), of nitroxide spin labels is dependent on the local environmental polarity. The dependence of a(o)(N) in fluid phospholipid bilayer membranes on the C-atom position, n, of the nitroxide in the sn-2 chain of a spin-labeled diacyl glycerophospholipid therefore determines the transmembrane polarity profile. The polarity variation in phospholipid membranes, with and without equimolar cholesterol, is characterized by a sigmoidal, trough-like profile of the form (1 + exp [(n - n(o))/lambda])(-1), where n = n(o) is the point of maximum gradient, or polarity midpoint, beyond which the free energy of permeation decreases linearly with n, on a characteristic length-scale, lambda. Integration over this profile yields a corresponding expression for the permeability barrier to polar solutes. For fluid membranes without cholesterol, n(o) approximately 8 and lambda approximately 0.5--1 CH(2) units, and the permeability barrier introduces an additional diffusive resistance that is equivalent to increasing the effective membrane thickness by 35--80%, depending on the lipid. For membranes containing equimolar cholesterol, n(o) approximately 9--10, and the total change in polarity is greater than for membranes without cholesterol, increasing the permeability barrier by a factor of 2, whereas the decay length remains similar. The permeation of oxygen into fluid lipid membranes (determined by spin-label relaxation enhancements) displays a profile similar to that of the transmembrane polarity but of opposite sense. For fluid membranes without cholesterol n(o) approximately 8 and lambda approximately 1 CH(2) units, also for oxygen. The permeation profile for polar paramagnetic ion complexes is closer to a single exponential decay, i.e., n(o) lies outside the acyl-chain region of the membrane. These results are relevant not only to the permeation of water and polar solutes into membranes and their permeabilities, but also to depth determinations of site-specifically spin-labeled protein residues by using paramagnetic relaxation agents.

Magnetic Resonance Spectroscopy↗
PubMed2001

Interaction of N-myristoyldimyristoylphosphatidylethanolamine with dimyristoylphosphatidylcholine investigated by differential scanning calorimetry: binary phase diagram.

The temperature-composition phase diagram was derived for hydrated, binary mixtures of N-myristoyldimyristoylphosphatidylethanolamine (N-14 DMPE) and dimyristoylphosphatidylcholine by high sensitivity differential scanning calorimetry. Gel phase immiscibility was detected in mixtures containing up to 20 mol% N-14 DMPE and there was no evidence for compound formation between the two components. In the fluid phase nearly complete miscibility is indicated by the calorimetric data. These results are relevant to understanding the role of N-acylphosphatidylethanolamines in the stress combating responses of organisms and in their application to developing liposome-based drug delivery systems.

Calorimetry, Differential Scanning↗