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Biomedical subjects

D Marsh

Publications and source records attributed to D Marsh.

At least 235 records · Page 13Linked to original sources

Right atrial thrombus formation screening using two-dimensional echocardiograms in neonates with central venous catheters.

Two-dimensional echocardiograms were used to prospectively screen 49 patients with 56 central venous catheters for right atrial thromboses from October 1985 to May 1986. All but four patients received a two-dimensional echocardiogram prior to insertion of the catheter. Once the catheters were in place, two-dimensional echocardiograms were performed no later than 3 weeks after insertion and then every 14 days until the catheter was removed. A single thrombus was detected 79 days after catheter placement (an incidence of 1.8%). Previous recommendations for weekly screening with two-dimensional echocardiogram were based on case reports alone. The 95% confidence limits for a negative two-dimensional echocardiogram result suggest that the initial two-dimensional echocardiogram screen for thrombus be obtained no sooner than 3 weeks after catheter insertion. In addition, significant gastrointestinal disease requiring operative intervention was present in 10 of 11 previous case reports as well as in our patient. Further studies with larger sample sizes are needed to determine whether subgroups of infants exist who are at a relatively higher risk for right atrial thrombus formation.

Catheterization, Peripheral↗

Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosinetriphosphatase in membranes from Squalus acanthias.

The sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)-ATPase] in membranous preparations from Squalus acanthias has been spin-labeled on sulfhydryl groups after prelabeling with N-ethylmaleimide. Saturation-transfer electron spin resonance spectroscopy has been used to study the rotational motions of the labeled protein on the microsecond time scale. Effective rotational correlation times deduced from the diagnostic line-height ratios in the second-harmonic, 90 degrees out-of-phase (V2') spectra are much larger than those deduced from the spectral integrals, indicating the presence of large-scale segmental motions, in addition to rotation of the protein as a whole. Experiments involving controlled cross-linking of the protein by glutaraldehyde, as well as measurements of the line broadening of the conventional electron spin resonance spectra, support this interpretation. Both the spectral integrals and diagnostic line-height ratios are found to increase irreversibly with time on incubation at temperatures greater than 20 degrees C, corresponding to a decrease in the segmental motion of the protein and probably also in the overall protein rotation. The native enzyme displays a marked nonlinearity in the Arrhenius temperature dependence of the activity at temperatures above 20 degrees C, and the activity decreases with a half-life of ca. 70 min on incubation at 37 degrees C (but not on incubation at low temperature), paralleling the time- and temperature-dependent changes in the saturation-transfer spectra of the labeled protein. Both of these observations suggest that the changes observed in the molecular dynamics could correspond to functional properties of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase.

The selectivity of interaction of various cardiolipin analogues with beef heart cytochrome oxidase in reconstituted complexes with dimyristoylphosphatidylcholine has been studied by electron spin resonance spectroscopy, using lipids spin-labeled in the acyl chains. No difference in selectivity is observed between cardiolipin and its monolyso derivative, and similarly no selectivity is observed between phosphatidylcholine and lysophosphatidylcholine. Removal of the cardiolipin charge by methylation of the phosphate groups reduces but does not eliminate selectivity relative to phosphatidylcholine. The dependence of the lipid selectivity on head group and chain composition is in the order cardiolipin approximately equal to monolysocardiolipin greater than acylcardiolipin greater than dimethylcardiolipin greater than phosphatidylcholine approximately equal to lysophosphatidylcholine, where acylcardiolipin has the spin-label chain attached at the center -OH of the head group. The degree of association of the negatively charged cardiolipin derivatives with cytochrome oxidase decreases with increasing salt concentration, to a level comparable to that for dimethylcardiolipin. At high ionic strength there is still a marked selectivity relative to phosphatidylcholine. Li+ ions are more effective in screening the interaction than are Na+ ions, and divalent ions are more effective than monovalent ions. The selectivity for cardiolipin is only slightly reduced on titrating the protein to high pH. Alkylation of the protein with N-ethylmaleimide has little effect on the titration behavior. Covalent modification of the protein by reaction with citraconic anhydride decreases the selectivity of interaction with cardiolipin. It is concluded that cardiolipin possesses an additional specificity of interaction with cytochrome oxidase other than that of purely electrostatic origin.

Animals↗

Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers.

The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain. The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated. At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively. The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine. The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane.

Coliphages↗

Molecular exchange at the lipid-rhodopsin interface: spin-label electron spin resonance studies of rhodopsin-dimyristoylphosphatidylcholine recombinants.

The photoreceptor protein rhodopsin has been reconstituted with a single phospholipid species, dimyristoylphosphatidylcholine, at a range of different lipid/protein ratios, and the exchange rate at the lipid-protein interface has been determined from the electron spin resonance spectra of spin-labeled phosphatidylcholine. For recombinants with lipid/protein ratios in the range 41:1 to 102:1 (mol/mol), the electron spin resonance spectra of 1-acyl-2-[14-(4,4-dimethyloxazolidine-N-oxyl)stearoyl]-sn-glycero-3- phosphocholine consist of a fluid component similar to that found in pure lipid bilayers and a motionally restricted component corresponding to lipids whose motion is reduced by interaction with the intramembranous surface of rhodopsin. The relative proportion of the motionally restricted component increases with increasing protein content in the complex. Spectral subtraction with fluid and motionally restricted components (from fluid- and gel-phase lipid, respectively), which best fit the apparent components in the complex, reveals that 22 +/- 2 lipids per 39,000-dalton protein are motionally restricted, independent of lipid/protein ratio and of temperature. Simulation of the two-component spectra with the exchanged-coupled Bloch equations gives values for both the fraction of motionally restricted component and the exchange rate between the two components. Using fixed motionally restricted and fluid component line shapes at a given temperature, it is possible to obtain a consistent description of the lipid/protein ratio dependence of the spectra at each temperature. The number of motionally restricted lipids obtained by simulation, allowing for exchange, is 23 +/- 3 per 39,000-dalton protein, again independent of temperature and of lipid/protein ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Head group and chain length dependence of phospholipid self-assembly studied by spin-label electron spin resonance.

The critical micelle concentrations (cmc's) of a variety of spin-labeled phospholipids, 1-acyl-2-[4-(4,4-dimethyloxazolidine-N-oxyl)valeryl]-sn-glycero-3-pho sph o derivatives, have been determined by electron spin resonance (ESR) spectroscopy. The narrow, three-line ESR spectra of the rapidly tumbling monomers are clearly distinguished from the spin-spin broadened spectra of the micellar aggregates, allowing a direct determination of the concentrations of the two species. The influence of both the hydrocarbon chain length and the polar head group on the energetics of self-assembly has been studied. For phosphatidylcholine, 1n [cmc] decreases linearly with the length of the sn-1 chain. The gradient of this linear dependence corresponds to a free energy of transfer of the monomer from the aqueous phase to the micelle of delta Gtr = -1.1RT per CH2 group. The cmc's of the 1-lauroyl derivatives of both phosphatidylcholine and phosphatidylglycerol have relatively shallow, biphasic temperature dependences with a minimum at approximately 20 degrees C. Both of these properties are characteristic of the hydrophobic effect, with the free energy of transfer being slightly less than that for the solubility of n-hydrocarbons in water, corresponding to the reduced configurational entropy of the lipid chains in the micellar state. The cmc's of the 1-lauroyl derivatives of the phospholipids in 0.15 M NaCl, for their various charge states, are as follows: phosphatidic acid(2-), 0.77 mM; phosphatidic acid(1-), 0.13 mM; phosphatidylserine(1-), 0.24 mM; phosphatidylglycerol(1-), 0.17 mM; phosphatidylcholine, 0.10 mM; phosphatidylethanolamine, 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Spin Resonance Spectroscopy↗

Lipid mobility and order in bovine rod outer segment disk membranes. A spin-label study of lipid-protein interactions.

Lipid-protein interactions in bovine rod outer segment disk membranes have been studied by using a series of eight stearic acid spin-label probes which were labeled at different carbon atom positions in the chain. In randomly oriented membrane dispersions, the electron spin resonance (ESR) spectra of the C-8, C-9, C-10, C-11, C-12, C-13, and C-14 atom positional isomers all apparently consist of two components. One of the components corresponds closely to the spectra obtained from dispersions of the extracted membrane lipids, and the other, which is characterized by a considerably greater degree of motional restriction of the lipid chains, is induced by the presence of the protein. Digital subtraction has been used to separate the two components. The proportion of the motionally restricted lipid component is approximately constant, independent of the position of the spin-label group, and corresponds to 30-40% of the total spin-label spectral intensity. The hyperfine splitting of the outer maxima in the difference spectra of the motionally restricted component decreases, and concomitantly, the line widths increase with increasing temperature but change relatively little with increasing distance of the spin-label group from the polar head-group region. This indicates that the corresponding chain motions of the protein-interacting lipids lie in the slow-motion regime of spin-label ESR spectroscopy (tau R approximately 10(-8) S) and that the mobility of these lipids increases with increasing temperature but does not vary greatly along the length of the chain. The data from the hyperfine splittings also suggest the existence of a polarity gradient immediately adjacent to the protein surface, as observed in the fluid lipid regions of the membrane. The more fluid lipid component is only slightly perturbed relative to the lipids alone (for label positions 5-14, inclusive), indicating the presence of chain motions on the nanosecond time scale, and the spectra also reveal a similar polarity profile in both lipid and membrane environments. ESR spectra have also been obtained as a function of magnetic field orientation with oriented membrane samples. For the C-14 atom positional isomer, the motionally restricted component is observed to have a large hyperfine splitting, with the magnetic field oriented both parallel and perpendicular to the membrane normal. This indicates that the motionally restricted lipid chains have a broad distribution of orientations at this label position.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Selectivity of lipid-protein interactions.

The spin label ESR and intrinsic fluorescence quenching methods of determining the selectivity of interactions of lipids with integral membrane proteins are summarized. The selectivity patterns of phospholipids, fatty acids, and steroids are reviewed for a variety of integral proteins. Where appropriate, correlations are established with biochemical assays of the effects of specific lipids on enzymatic activity and transport function.

Electron Spin Resonance Spectroscopy↗

Integral membrane proteins significantly decrease the molecular motion in lipid bilayers: a deuteron NMR relaxation study of membranes containing myelin proteolipid apoprotein.

The influence of the myelin proteolipid apoprotein on lipid chain order and dynamics was studied by 2H NMR of membranes reconstituted with specifically deuterated dimyristoyl phosphatidylcholines. Quadrupolar echo and saturation recovery experiments were fitted by numerical solution of the stochastic Liouville equation, using a model that includes both inter- and intramolecular motions [Meier, P., Ohmes, E. & Kothe, G. (1986) J. Chem. Phys. 85, 3598-3614]. Combined simulations of both the relaxation times and the quadrupolar echo line shapes as a function of pulse spacing allowed unambiguous assignment of the various motional modes and a consistent interpretation of data from lipids labeled on the C-6, C-13, and C-14 positions of the sn-2 chain. In the fluid phase, the protein has little influence on either the chain order or the population of gauche rotational isomers but strongly retards the chain dynamics. For 1-myristoyl-2-[13-2H2] myristoyl-sn-glycero-3-phosphocholine at 35 degrees C, the correlation time for chain fluctuation increases from 20 nsec to 650 nsec and for chain rotation from 10 nsec to 180 nsec, and the gauche isomer lifetime increases from 0.15 nsec to 1.75 nsec, on going from the lipid alone to a recombinant of protein/lipid ratio 0.073 mol/mol. The results are essentially consistent with spin-label ESR studies on the same system [Brophy, P.J., Horvath, L.I. & Marsh, D. (1984) Biochemistry 23, 860-865], when allowance is made for the different time scales of the two spectroscopies.

Animals↗

Does the use of the operating microscope improve the results of peripheral nerve suture?

Twenty-four cases of complete division of median or ulnar nerves were assessed on two occasions after direct suture, with an interval of three years between assessments. Clinical, electrophysiological and timed functional tests were used. All the operations had been performed by one surgeon, using the operating microscope for approximately half the cases but not for the other half. The results were analysed, and the patient's age, any delay between injury and suture, and the duration of follow-up were all found to exert strong and consistent effects on the scores obtained. After controlling for these factors, there was no consistent difference between the results of the two surgical methods. It is concluded that, at least in the hands of one particular surgeon, the use of the operating microscope gives no better results than careful epineural suture performed without it.

Adult↗

Three years after Colles' fracture. A prospective review.

A prospective study was made over a three-year period of 900 consecutive unilateral Colles' fractures. The radiographic features at the time of fracture, after reduction and one week later were measured and correlated with grip strength and range of movement at three years. The most significant radiographic feature to influence the outcome was the presence of shortening of the radius one week after reduction of the fracture. Persistent dorsal tilt, radiocarpal joint involvement and ulnar styloid fracture were each associated with reduced range of movement, but had no effect on grip strength. Extension of the fracture into the distal radio-ulnar joint was associated with reduced grip strength but had no effect on range of movement. Radial tilt of the radial fragment did not correlate with any aspect of the result after three years.

Aged↗

[Correlation between the atmospheric level of antigen Amb-al (AgE) and the number of Ambrosia artemisiaefolia pollen grains in Lyon and neighboring regions].

Accurate quantitation of the atmospheric levels of pollens and other allergenic particulate is laborious and may not always correlate with the concentration of allergen within the particulate. This study examined the atmospheric contamination by short ragweed (Ambrosia artemisiae folia) over 7 weekly periods during ragweed pollination in 6 localities of France centered around Lyon. The correlation between data obtained from the conventional microscopic numeration of pollen grain and and the quantitation of the allergen Amb-al level by radioimmunoassay was investigated. For the total of 41 paired analyses of the actual numbers of ragweed pollen grains and computed numbers of pollen grains from the Amb-al levels there was an excellent overall correlation (r = 0.90, p 0.001) thus the radioimmunoassay of Amb-al represent a simple and relevant alternative to microscopic numeration of pollen grains.

Air Pollutants↗

Prediction of the critical micelle concentrations of mono- and di-acyl phospholipids.

The experimental linear chainlength dependence of the logarithm of the critical micelle concentration (ln [CMC]) of mono- and di-acyl phosphatidylcholines, together with recent measurements of the polar headgroup dependence of the CMCs of a variety of spin-labelled phospholipids, and the relative solubilities of saturated and unsaturated hydrocarbons, have been combined to devise a scheme for making general predictions of the CMCs of both mono- and di-acyl phospholipids, such as are found in biological membranes.

Liposomes↗

Free volume model for lipid lateral diffusion coefficients. Assessment of the temperature dependence in phosphatidylcholine and phosphatidylethanolamine bilayers.

The lipid lateral diffusion coefficients, DT, in fluid-phase phosphatidylcholine and phosphatidylethanolamine bilayers have been analysed in terms of the free-volume diffusion model by fitting the expression: DT = AT exp[- B/(T - T0)] to the observed temperature dependence, where A, B and T0 are the parameters to be optimized. Application of an unconstrained optimization procedure to data obtained from excimer formation (Galla et al. (1979) J. Membrane Biol. 48, 215-236) and from fluorescence photobleaching (Vaz et al. (1985) Biochemistry 24, 781-786) provides statistical evidence for a free-volume model as opposed to a simple Stokes-Einstein model (T0 = 0), only in certain cases. In the instances for which the parameter T0 can be determined with a reasonable degree of accuracy, it is found that this characteristic temperature at which the free volume extrapolates to zero lies below the bilayer gel-to-fluid phase transition temperature and does not coincide with the pre-transition temperature for phosphatidylcholines.

Diffusion↗

Preferential association of apocytochrome c with negatively charged phospholipids in mixed model membranes.

The mitochondrial precursor protein, apocytochrome c, binds to model membranes containing negatively charged phospholipids (Rietveld, A., Sijens, R., Verkleij, A.J. and Kruijff, B. (1983) EMBO J. 2, 907-913). In the present paper the effect of apocytochrome c on the lipid distribution in model membranes, consisting of neutral and acidic phospholipids, is examined. Both ESR and fluorescence energy transfer experiments show that the protein preferentially interacts with the negatively charged phospholipid in the mixed model membranes. Semi-quantitative analysis of the fluorescence energy transfer from the single tryptophan in apocytochrome c to the parinaric acid in phosphatidylserine or phosphatidylcholine in mixed bovine brain phosphatidylserine/egg phosphatidylcholine vesicles reveals and average donor-acceptor distance of 22-26 A and 26-30 A for phosphatidylserine and phosphatidylcholine, respectively. In addition, these experiments demonstrate that this preferential interaction does not induce the separation of large domains enriched in complexes of apocytochrome c with negatively charged phospholipids and domains enriched in neutral lipids.

Apoproteins↗

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance.

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Preparation and high-performance size-exclusion chromatographic (HPSEC) analysis of fluorescein isothiocyanate-hydroxyethyl starch: macromolecular probes of the blood-lymph barrier.

A procedure is described for the preparation of fluorescein isothiocyanate-labeled hydroxyethyl starch (FITC-HES). Chromatographic techniques for the purification and analysis of FITC-HES that include the development of a high-performance size-exclusion chromatographic (HPSEC) technique with a fluorescence spectrophotometer-computer detection system are described. FITC-HES macromolecules have a wide range of hydrodynamic radii (ae greater than 120- less than 10 A) and the substitution ratio of FITC to the size-selected HES macromolecules remains constant throughout the chromatographic range. The predominant isoelectric point (pI) of the multiple acidic isomers of FITC-HES is 4.6. In vitro, the very large FITC-HES macromolecules (greater than 100 A) are rapidly degraded (15 sec) by alpha-amylase in control dog plasma. While most of the large molecules (100-20 A) remain intact for greater than 24 hr, this degradation is not associated with the loss of FITC from HES. In vivo, the rate of this reaction appeared to be accelerated and the degradation of FITC-HES between 0.1 and 12 hr was small. Illustration of the HPSEC quantitation of the spectrum of FITC-HES macromolecules in "near steady-state" lymph (L) and plasma (P) samples and the L/P ratio show that this technique can be used to describe the size selectivity of the blood-lymph barrier under conditions of unaltered capillary pressure. We propose that the size-selected solvent-drag reflection coefficient (sigma f) curves for the anionic FITC-HES under conditions of elevated capillary pressure is a measure of macromolecular convective permeability of the blood-lymph barrier.

Animals↗