A new aesthesiometer for testing sensibility in the hand.
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Carpal Tunnel Syndrome↗
Biomedical subjects
D Marsh
Publications and source records attributed to D Marsh.
Investigations on the insertion of the mitochondrial precursor protein apocytochrome c into model membranes.
Different aspects of the interaction of apocytochrome c and model membranes composed of negatively charged lipids, were studied in order to get insight into the nature of this interaction. The effect of the protein on the lipid packing properties are revealed by DSC, ESR and monolayer techniques. These experiments clearly demonstrate that upon electrostatic interaction with the negatively charged phospholipids, apocytochrome c is able to penetrate into the hydrophobic region of the model membrane. In the case of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, this results in a perturbation of 160 lipid molecules per apocytochrome c molecule. Most likely, apocytochrome c disrupts the formation of the gel phase and restricts the lipid chain motion above the gel to liquid-crystalline phase transition. Tryptophan fluorescence measurements confirm that at least a part of the protein penetrates into the bilayer, and suggest that after this penetration, the tryptophan (residue no. 59) is located in the glycerol backbone region of the phospholipids. Although the secondary structure of apocytochrome c is predicted to contain about 35% of alpha-helical structure, the CD pattern of an aqueous solution of the protein is featureless. However, negatively charged lipids are able to express this alpha-helical potency in the apocytochrome c, which might be important for the insertion of the protein into lipid membranes.
Animals↗
Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model.
The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)
Cyclic N-Oxides↗
Spin-label studies on the origin of the specificity of lipid-protein interactions in Na+,K+-ATPase membranes from Squalus acanthias.
The pH dependence and salt dependence of the lipid-protein interactions of phosphatidic acid, phosphatidylserine, and stearic acid with Na+,K+-ATPase membranes from Squalus acanthias have been studied with spin-label electron spin resonance spectroscopy, using lipids with nitroxide labels on the 14-position C atom of the sn-2 chain. For phosphatidic acid and stearic acid, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 6.6 and 8.0, respectively. In contrast, the pKa of stearic acid in the bulk lipid environment of the membrane is estimated from spin-label spectroscopy to be approximately equal to 6.6. The fraction of motionally restricted phosphatidylserine spin-label remains constant over the pH range 4.7-9.2. In the fully dissociated state the fractions of motionally restricted spin-labeled phosphatidic and stearic acids decrease with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M. For stearic acid the net decrease is comparable to that obtained on protonation, but for phosphatidic acid the decrease is considerably smaller (by approximately 55%) than that obtained on protonating the lipid. The fraction of motionally restricted phosphatidylserine spin-label varies relatively little with salt concentration up to 1 M NaCl. Direct electrostatic effects alone cannot account for the whole of the observed specificity of interaction of the two phospholipids with Na+,K+-ATPase membranes.
Animals↗
Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites.
The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.
Animals↗
Polymorphic phase behavior of cardiolipin derivatives studied by 31P NMR and X-ray diffraction.
The polymorphic phase behavior of cardiolipin (diphosphatidylglycerol) analogues with two to five chains per phospholipid head group, namely, dilysocardiolipin, monolysocardiolipin, cardiolipin, and acylcardiolipin, respectively, has been studied by 31P NMR and X-ray diffraction. Dilysocardiolipin dispersions at low salt concentration are micellar, and a transition to a lamellar phase takes place between 1 and 2 M NaCl. From light-scattering measurements, it is also found that a transition takes place from the micellar state with a midpoint at 5.2 mM CaCl2, 0.95 M HClO4, and 1.5 M NaCl. Monolysocardiolipin dispersions are lamellar throughout the concentration range from zero to saturated NaCl. Cardiolipin dispersions undergo a transition from a lamellar to an inverted hexagonal phase between 1 and 2 M NaCl. Acylcardiolipin dispersions are in an inverted hexagonal phase throughout the concentration range from zero to saturated NaCl. The chemical shift anisotropies of both phosphate groups in dilysocardiolipin and of one of the phosphate groups in monolysocardiolipin are drastically reduced in the lamellar phase, indicating a different conformation of the phosphatidyl head group from that normally found in diacyl phospholipid bilayers. The results provide strong support for the "shape" concept of lipid polymorphism when viewed in its most general form including configurational entropy, hydrophobic effects, etc. and indicate the importance of head-group interactions in determining the lipid phase behavior.
Animals↗
Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels.
Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.
Animals↗
Spin-label studies of lipid-protein interactions in (Na+,K+)-ATPase membranes from rectal glands of Squalus acanthias.
Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from the rectal gland of Squalus acanthias have been studied by using spin-labeled lipids in conjunction with electron spin resonance (ESR) spectroscopy. Lipid-protein associations are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component which corresponds very closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled lipids whose motion is very significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A stoichiometry of approximately 66 lipids per 265 000-dalton protein is found for the motionally restricted component of those spin-labeled lipids (e.g., phosphatidylcholine) which show least specificity for the protein. This corresponds approximately to the number of lipids which may be accommodated within the first shell around the alpha 2 beta 2 protein dimer. A selectivity of the various spin-labeled lipids for the motionally restricted component associated with the protein is found in the following order: cardiolipin greater than phosphatidylserine approximately stearic acid greater than or equal to phosphatidic acid greater than phosphatidylglycerol approximately phosphatidylcholine approximately phosphatidylethanolamine approximately androstanol.
Animals↗
Hydration of noncharged lipid bilayer membranes. Theory and experiments with phosphatidylethanolamines.
Calorimetric measurements have been made on the thermodynamics of the chain-melting phase transition of saturated diacylphosphatidylethanolamines, with chains containing 12-20 carbons, as a function of water content. The transition temperature, Tt, and the transition enthalpy, and entropy all decrease with an increase in water content; however, the effect on Tt lessens with an increase in chainlength. These results are compared with a theoretical description of lipid hydration in terms of the interlamellar water polarization (i.e., modified water structure) in the interbilayer region. The measured free energy, enthalpy and entropy of the transition and the transition temperature have an approximate hyperbolic tangent dependence on water content, infinity tanh (dw/2 xi), where dw is the interlamellar water-layer thickness and xi approximately equal to 0.25 nm is the water-order correlation length, in agreement with the theory. Auxiliary x-ray diffraction experiments yield results on the repulsive hydration forces between lipid lamellae consistent with the theory, and allow an estimate of the water orienting potential of the interface. The molecular origin of this potential is discussed in electrostatic terms, and the values of its associated molecular parameters are found to yield the right size of hydrational thermodynamic quantities. The theory thus provides an integrated, clear, and simple approach to the hydration properties of lipid membranes.
Biophysical Phenomena↗
Role of prostaglandins and angiotensin II in experimental glomerulonephritis.
Moderate autologous nephrotoxic serum nephritis (NSN) in rats causes no reduction in GFR despite a reduction in ultrafiltration coefficient (Kf) to less than one-half of normal. An increase in intraglomerular hydraulic pressure maintains GFR, but the signal and efferent mechanisms for this adaptation remain unknown. Indomethacin and saralasin were used to study the possible roles of prostaglandins and angiotensin II (A-II) in the adaptation to NSN. Indomethacin decreased renal blood flow (RBF) in NSN (-8.6%, P less than 0.001), but not in controls. Renal vascular resistance (RVR) increased in NSN (+ 9.6%, P less than 0.01), but decreased in controls (-5.6%, P less than 0.01). GFR decreased in NSN (-22.3%, P less than 0.01), but increased in controls (+ 10.3%, P less than 0.001). Urinary PGE2 excretion decreased markedly both in NSN and controls. With combined treatment using indomethacin and saralasin, RBF increased in NSN (+ 22.4%, P less than 0.001), but did not change in controls. RVR decreased in NSN (-21.5%, P less than 0.001), but was unchanged in controls. GFR remained unchanged both in NSN and controls. With saralasin alone, RBF increased both in NSN (+ 21.4%, P less than 0.001) and in controls (+ 14.4%, P less than 0.001). RVR decreased both in NSN (-21.8%, P less than 0.001) and controls (-18.7% P less than 0.001). GFR increased (+ 12.3%, P less than 0.05) in NSN, but did not change in controls. Urinary PGE2 excretion was increased in NSN compared to controls, decreased markedly in NSN after either indomethacin or saralasin infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Angiotensin II↗
The renal hemodynamic consequences of experimental glomerulonephritis.
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Animals↗
X-ray diffraction study of the polymorphism of hydrated diacyl- and dialkylphosphatidylethanolamines.
The structure and polymorphism of a homologous series of diacyl- and of dialkylphosphatidylethanolamines have been investigated by X-ray diffraction, calorimetry, and density measurement. The compositional dependence of the repeat spacings of the gel (L beta or L beta'), fluid bilayer (L alpha), and inverted hexagonal (HII) phases has been determined both for the short chain length (di-C12) dialkyl didodecylphosphatidylethanolamine (DDPE) and for the long chain length (di-C20) diacyl diarachinoylphosphatidylethanolamine (DAPE). These data, in conjunction with the measured phase transition temperatures obtained both by X-ray diffraction and by differential scanning calorimetry, have been used to construct phase diagrams for the two lipids. DDPE exhibits metastable behavior in the L beta and L alpha phases below 44 degrees C at all water contents and forms cubic and other nonlamellar phases between the L alpha and HII phases. At low water contents, crystalline and fluid phases coexist at temperatures up to 83 degrees C. For DAPE, the behavior is simpler. In the gel phase, the hydrocarbon chains are tilted at 29 degrees to the bilayer normal, and metastability is only observed at water contents below 3 wt %. The L alpha phase is adopted within a narrow temperature range and then transforms directly to the HII phase. The structural parameters of the L beta (L beta'), L alpha, and HII phases of DDPE and DAPE have been calculated from the X-ray data, in conjunction with the measured values of lipid partial specific volume. In addition, the chain-length dependence of the repeat spacings of the phases has been measured for the homologous series of diacyl and dialkyl lipids. Taken together, the results allow a detailed description of the effects of temperature, hydration, and chain length on the polymorphism of the saturated phosphatidylethanolamines.
Calorimetry↗
Stoichiometry and specificity of lipid-protein interaction with myelin proteolipid protein studied by spin-label electron spin resonance.
The interaction of spin-labeled lipids with the myelin proteolipid apoprotein in complexes with dimyristoylphosphatidylcholine of varying lipid/protein ratios has been studied with electron spin resonance spectroscopy. A first shell of approximately 10 lipids per 25 000-dalton protein is found to be motionally restricted by the protein interface. This stoichiometry is consistent with a hexameric arrangement of the protein in the membrane. A selectivity of the various spin-labeled lipids for the motionally restricted component at the protein interface is found in the order stearic acid greater than phosphatidic acid greater than cardiolipin approximately greater than phosphatidylserine greater than phosphatidylglycerol approximately equal to phosphatidylcholine greater than phosphatidylethanolamine greater than androstanol approximately greater than cholestane.
Animals↗
An immunological study of rat acetylcholinesterase: comparison with acetylcholinesterases from other vertebrates.
We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse greater than human greater than bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., 1 M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and detergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer G4. Although we cannot exclude the existence of significant differences between the various molecular forms of acetylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by posttranslational modifications.
Acetylcholinesterase↗
Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study.
The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.
Animals↗
Dynamic structure and phase behavior of dimyristoylphosphatidylethanolamine bilayers studied by deuterium nuclear magnetic resonance.
The dynamic structure of dimyristoylphosphatidylethanolamine bilayers has been studied by deuterium nuclear magnetic resonance spectroscopy of the perdeuterated sn-2 chain. The order parameter profile of the lipid chains in the fluid phase is qualitatively similar to that found for other phospholipids, but the order parameter plateau is ca. 15% higher than found for dimyristoylphosphatidylcholine at a comparable reduced temperature. The chains of dimyristoylphosphatidylethanolamine undergo a segmental motion in the gel phase, which for segments close to the end of the chain approximates continuous axial diffusion. In the phase-transition region, spectra are observed that can be best described in terms of the interconversion of coexisting lipid phases through the transition.
Kinetics↗
Metastability and polymorphism in the gel and fluid bilayer phases of dilauroylphosphatidylethanolamine. Two crystalline forms in excess water.
The phase behavior of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) in excess water has been studied by differential scanning calorimetry and x-ray diffraction. In addition to the usual gel phase L beta of lamellar periodicity 5.07 nm and the fluid bilayer phase L alpha of periodicity 4.5 nm, two distinct crystalline forms may also be spontaneously adopted. The L beta phase is only produced by cooling from L alpha and is metastable, relaxing to one of the crystalline forms on incubation. One crystalline polymorph, designated beta 2, has a lamellar periodicity of 4.55 nm and corresponds to the structure of DLPE crystallized from a variety of organic solvents. The other crystalline polymorph, designated beta 1, has a lamellar periodicity of 3.78 nm, which implies that in this form the hydrocarbon chains are tilted at approximately 40 degrees to the bilayer normal. The beta 2 polymorph is obtained on dispersing crystalline DLPE directly in water at T less than 43 degrees C, on incubation in the L alpha phase at 30 degrees C less than T less than 43 degrees C, or on heating the beta 1 form slowly to T greater than 35 degrees C. The beta 1 polymorph is obtained on incubating the L beta phase at T less than 30 degrees C. By calorimetry, the L beta phase undergoes an endothermic transition (delta H = 15.5 kJ X mol-1 (3.7 kcal X mol-1)) at 30.6 degrees C to the fluid bilayer phase L alpha. The beta 1 phase undergoes an endothermic transition (delta H congruent to 50 kJ X mol-1 (12 kcal X mol-1)) to L alpha at 35 degrees C. The beta 2 phase undergoes an endothermic transition (delta H = 57 kJ X mol-1 (13.7 kcal X mol-1)) to L alpha at 43 degrees C.
Drug Stability↗
Calorimetric studies of the gel-fluid (L beta-L alpha) and lamellar-inverted hexagonal (L alpha-HII) phase transitions in dialkyl- and diacylphosphatidylethanolamines.
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Calorimetry, Differential Scanning↗