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Biomedical subjects

D Marsh

Publications and source records attributed to D Marsh.

At least 91 records · Page 5Linked to original sources
PubMed1998

Concepts of fracture union, delayed union, and nonunion.

Laboratory and clinical scientists and practicing clinicians need definitions of union, delayed union, and nonunion. Fracture union is a gradual process, so quantitative measures are the most meaningful. However, end point definitions also are useful, but they need empirical validation. The measure that has received the best validation in human fractures is bending stiffness. Quantitative radiologic assessment of healing is difficult because varying patterns of bone bridging can occur, including periosteal, endosteal, and intercortical patterns. Natural fracture healing was studied in 43 cases of isolated, closed, conservatively treated tibial shaft fracture with serial measurements of bending stiffness and standard radiographs. Three healing groups were defined on the basis of stiffness recovery patterns. Four cases showed delayed union, defined as failure to reach a stiffness of 7 N-m per degree by 20 weeks from fracture. The remaining cases had normal union, but at differing rates. Callus index was used as a measure of periosteal new bone formation. Stiffness measurements correlated more strongly than callus index with injury severity and functional outcome at 6 months. However, the callus index predicted behavior in those fractures that showed no tendency to heal at the 10-week stage. That is, absence of periosteal new bone in these cases presaged delayed union. These delayed union cases all eventually healed, still without producing periosteal callus, but other fractures in the series healed very rapidly, also without periosteal callus. The implication is that endosteal healing is capable of very rapid fracture bridging if conditions are right, but it also can occur late, after the periosteal healing response has ceased. These observations suggest a more rational approach to the definition of union, delayed union, and nonunion than that provided by the selection of arbitrary times. For conservatively treated fractures at least, delayed union can be defined as the cessation of the periosteal response before the fracture successfully has been bridged. Nonunion is the cessation of both the periosteal and endosteal healing responses without bridging.

Animals↗
PubMed1997

Characterization of the secondary structure and assembly of the transmembrane domains of trypsinized Na,K-ATPase by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has been used to compare native Na,K-ATPase-containing membranes with those trypsinized in the presence of either Rb+ or Na+ ions to remove the extramembranous parts of the protein. The protein secondary structure content deduced from the amide I band is approximately 30-35% alpha-helix, 37-40% beta-structure, and 13-15% random coil for native membranes from shark rectal gland and from pig kidney, in both the Na- and K-forms. Trypsinization in either Rb+ (a K+ congener) or Na+ removes approximately 35% of the amide I band intensity of native membranes from shark rectal gland. The protein secondary structural content of the trypsinized membranes lies in the range of approximately 23-32% alpha-helix, 37-46% beta-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuterium-exchanged alpha-helices, and between the component bands attributed to beta-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated alpha-helix and a broader range of frequencies for beta-structure. The kinetics of deuteration of the slowly exchanging population of protein amide groups is also changed on trypsinization. The mean rate constant for deuteration of trypsinized membranes is approximately half that for native membranes, whereas the proportion of amides contributing to this population increases on trypsinization. The temperature dependence of the amide I band in the Fourier transform infrared spectra indicates that the onset of thermal denaturation occurs at 58 degrees C for native membranes (in either Na+ or K+) and for membranes trypsinized in Rb+, but the major denaturation event for membranes trypsinized in Na+ occurs at approximately 84 degrees C. These results correlate with the functional properties of the intramembranous section of the enzyme.

Animals↗
PubMed1997

Membrane location of spin-labeled M13 major coat protein mutants determined by paramagnetic relaxation agents.

Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. These were used for site-directed spin-labeling to determine the location and assembly of the major coat protein incorporated in bilayer membranes of dioleoylphosphatidylcholine. The membrane location of the spin-labeled cysteine residues was studied with molecular oxygen and Ni2+ ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-saturation electron spin resonance (ESR) spectroscopy. The section of the protein around Thr36 is situated at the center of the membrane. Residue Thr46 is placed at the membrane surface in the phospholipid head group region with a short C-terminal section, including Ala49, extending into the aqueous phase. Residue Ala25 is then positioned consistently in the head group region of the apposing lipid monolayer leaflet. These positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydrophobic section (residues 25-46), and then drop abruptly in the aqueous phase (residue 49). Additionally, the strong immobilization and low oxygen accessibility of residue 25 are attributed to steric restriction at the hinge region between the transmembrane and N-terminal amphipathic helices. Sequence-specific modulations of the ESR parameters are also observed. Relatively low oxygen accessibilities in the hydrophobic region suggest intermolecular associations of the transmembrane helices, in agreement with saturation transfer ESR studies of the overall protein mobility. Relaxation enhancements additionally reveal a Ni2+ binding site in the N-terminal domain that is consistent with a surface orientation of the amphipathic helix.

Binding Sites↗
PubMed1997

Phosphatidylcholine-fatty acid membranes: effects of headgroup hydration on the phase behaviour and structural parameters of the gel and inverse hexagonal (H(II)) phases.

The phase behaviour and structural parameters of a homologous series of saturated diacyl phosphatidylcholine/fatty acid 1:2 (mol/mol) mixtures having chain lengths from C12 to C20 were studied by X-ray diffraction and calorimetry, as a function of water content. The chain-melting transition temperatures of the 1:2 PC/FA mixtures are found to be largely independent of the degree of hydration. For all chain lengths, the tilted L(beta') and rippled P(beta') gel phases of the pure PC component are replaced by an untilted L(beta) gel phase in the 1:2 PC/FA mixtures. This gel phase swells considerably upon hydration, with a limiting water layer thickness in the range 18-24 A, depending on the chain length. However, unlike pure phospholipid systems, the lateral chain packing within the gel phase bilayers is essentially identical in both the dry and the fully hydrated states. The fluid bilayer L(alpha) phase is suppressed in the 1:2 mixtures, being replaced by inverse non-lamellar phases for all chain lengths greater than C12, and at all levels of hydration. For chain lengths of C16 and greater, the inverse hexagonal H(II) phase is formed directly upon chain melting, at all water contents. For the shorter chain length mixtures, the behaviour is more complex, with the H(II) phase forming at low hydration, but with bicontinuous cubic phases appearing at higher levels of hydration. The implications of these surprising results are explored, in terms of the effective hydrophilicity of the associated PC and FA headgroups and the packing within the interfacial region. We suggest that the presence of the fatty acids significantly alters the lateral stress profile across the lipid monolayer in the fluid state, compared to that of the corresponding pure PC system, such that inverse phases, where the interface bends towards the water, become strongly favoured. Furthermore, for short chain lengths, packing constraints favour the formation of phases with negative interfacial Gaussian curvature, such as the bicontinuous cubic phases, rather than the H(II) phase, which has more severe chain packing frustration.

Calorimetry↗
PubMed1997

Spin-label studies on the anchoring and lipid-protein interactions of avidin with N-biotinylphosphatidylethanolamines in lipid bilayer membranes.

The specific binding of hen egg white avidin to phosphatidylcholine lipid membranes containing spin-labeled N-biotinylphosphatidylethanolamines (biotin-PESLs) was investigated by using ESR spectroscopy. Spin-labeled biotin-PEs were prepared with the nitroxide group at position C-5, C-8, C-10, C-12, or C-14 of the sn-2 chain and were incorporated at 1 mol % in lipid bilayer membranes of dimyristoylphosphatidylcholine. Binding of avidin produced a strong and selective restriction of the biotin-PE lipid mobility at all positions of chain labeling, as shown by the ESR spectra recorded in the fluid lipid phase. The spectral components of the fraction of the biotin-PESLs that were not complexed by avidin indicated that the mobility of the bulk membrane lipids was unperturbed by binding avidin, as demonstrated by difference spectroscopy. Comparison of the positional profiles and temperature dependences of the outer hyperfine splittings from the biotin-PESLs suggests that the C-12 and C-14 positions of the avidin-bound biotin-PEs are in register with the C-5 and C-7/C-6 positions, respectively, of the chains of the bulk membrane lipids. This indicates that the biotin-PEs are partially withdrawn from the membrane, with a vertical displacement of ca. 7-8 A, on complexation with avidin. In addition, the specific lipid-protein interaction with avidin results in a selective reduction in the rates of lipid chain motion, as shown by the increased ESR line widths. These data define the way in which avidin is anchored to lipid membranes containing biotin-PEs.

Avidin↗
PubMed1997

Lipid mixing in dimyristoyl phosphatidylcholine-dimyristoyl glycerol dispersions: spin label ESR studies.

The lipid transfer and mixing properties in hydrated dispersions of dimyristoyl phosphatidylcholine (DMPC)/dimyristoyl glycerol (DMG) binary mixtures have been investigated by using electron spin resonance spectroscopy of the spin-labelled lipid components. The assay system is based on the reduction in spectral broadening from spin-spin interactions that takes place by dilution of the spin-labelled lipid on transfer to a 9-fold excess of dispersions that contain no spin label. Lipid dispersions with DMPC:DMG compositions of 70:30, 40:60 and 20:80 mol/mol, for which the fluid phases have lamellar, inverted hexagonal and isotropic structures, respectively, have been studied. Essentially no transfer of spin-labelled lipid takes place for any of the lipid mixtures in the lamellar gel phase, or in dispersions of DMPC alone at all temperatures studied. The greatest degrees of transfer are found in the fluid phase of the DMPC/DMG mixtures. In general, the extent of lipid transfer is greater for the diacylglycerol component than for the phosphatidylcholine component. The extent of transfer of phosphatidylcholine is very low in the fluid lamellar phase of the 70:30 mol/mol DMPC/DMG, as compared with that of diacylglycerol. Only in the case of the 40:60 mol/mol DMPC/DMG mixture, in the inverted hexagonal phase, are the extents of transfer comparable for both phosphatidylcholine and diacylglycerol components, indicating a bulk transfer of lipid within the dispersions. The largest extent of transfer is found for diacylglycerol in the 20:80 mol/mol DMPC/DMG mixture in the isotropic phase.

Diglycerides↗
PubMed1997

Renormalization of the tension and area expansion modulus in fluid membranes.

Renormalization of the membrane tension and elastic area expansion modulus by thermally induced bending fluctuations is treated in terms of the formalism of Brochard, De Gennes, and Pfeuty (J. de Phys. (France). 37:1099-1104, 1976). The dependence of the renormalized tension on the bare membrane tension parallels the dependence on the fractional area extension of giant vesicles found experimentally by Evans and Rawicz (Physiol. Rev. Lett. 64:2094-2097, 1990), and suggests conditions for molecular dynamics simulations with membrane patches of limited size that might best represent the properties of macroscopic vesicles.

Elasticity↗
PubMed1997

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.

Electron Spin Resonance Spectroscopy↗
PubMed1997

Differential scanning calorimetry of chain-melting phase transitions of N-acylphosphatidylethanolamines.

Phosphatidylethanolamines in which the polar headgroup is N-acylated by a long-chain fatty acid (N-acyl PEs) are present in many plasma membranes under normal conditions, and their content increases dramatically in response to membrane stress in a variety of organisms. The thermotropic phase behavior of a homologous series of saturated N-acyl PEs, in which the length of the N-acyl chain is equal to that of the O-acyl chains attached at the glycerol backbone, has been investigated by differential scanning calorimetry (DSC). All fully hydrated N-acyl PEs with even chain lengths from C-12 to C-18 exhibit sharp endothermic chain-melting phase transitions in the absence of salt and in 1 M NaCl. Cooperative chain-melting is demonstrated directly by the temperature dependence of the electron spin resonance spectra from probe phospholipids bearing a spin label group in the acyl chain. The calorimetric transition enthalpy and the transition entropy obtained from DSC depend approximately linearly on the chain length with incremental values per CH2 group that exceed those of normal diacyl phosphatidylethanolamines, but to an extent that underrepresents the additional N-acyl chain. A thermodynamic model is constructed for the chain-length dependences and end effects of the calorimetric quantities, which includes a deficit proportional to the difference in O-acyl and N-acyl chain lengths for nonmatched chains, as is found and justified structurally for mixed-chain diacyl phospholipids. From data on the chain-length dependence of N-acyl diC16PEs, it is then deduced that the N-acyl chains are less well packed than the O-acyl chains and, from the data on the matched-chain N-acyl PEs, that the O-acyl chain packing is similar to that in normal diacyl PEs. The gel-to-fluid phase transition temperatures of the N-acyl PEs in the absence of salt are practically the same as those of the normal diacyl PEs of the corresponding chain lengths, although the transition enthalpies and entropies are appreciably greater, indicating entropy-enthalpy compensation. In 1 M NaCl, the transition temperatures are 3-4.5 degrees higher than in the absence of salt, representing the contribution of the electrostatic surface potential of the N-acyl PEs.

Acylation↗
PubMed1997

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies.

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.

Amino Acid Sequence↗
PubMed1997

Interaction of bee venom melittin with zwitterionic and negatively charged phospholipid bilayers: a spin-label electron spin resonance study.

Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phosphatidylserine, phosphatidylglycerol, and stearic acid) and zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) had the largest outer splittings in the presence of melittin. Neutral lipids (protonated stearic acid and diacylglycerol) displayed the largest increase in outer splitting on binding melittin, which was attributed to a change in the vertical location of these lipids in the bilayer. Both effects were more pronounced in DTPG than in DMPC.

Amino Acid Sequence↗
PubMed1997

Fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin.

The effect of bacteriorhodopsin (BR) on the percolation properties of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine bilayers was examined by studying the quenching of a lipid-bound fluorophore by a lipid-bound quencher, and by spin-spin interactions of a nitroxide-labeled lipid using electron spin resonance (ESR). At the low concentrations of BR used, differential scanning calorimetry showed that although the transition enthalpy was reduced in a concentration-dependent manner by incorporation of BR, the solidus and fluidus phase boundaries and overall shape of the heat capacity profiles were essentially unchanged. However, fluorescence quenching and spin-label ESR data showed that the domain topology, as reflected in the percolation properties, is strongly affected by the protein. In contrast to our previous fluorescence data for the pure lipid mixtures, quenching in the coexistence region is independent of the fluid phase fraction when BR is present. In addition, the percolation threshold estimated by spin-label ESR is shifted in the presence of BR to a higher gel phase fraction at a given lipid composition. Both the fluorescence quenching and spin-label ESR data, together with the results of earlier simulations, strongly suggest that the fluid phase domains are substantially larger and/or less ramified in the presence of BR than in its absence. We have previously reported a similar effect of a transmembrane peptide, pOmpA (Escherichia coli outer membrane protein A signal peptide), on fluid domain connectivity in binary phosphatidylcholine mixtures.

Bacteriorhodopsins↗
PubMed1997

Dichroic ratios in polarized Fourier transform infrared for nonaxial symmetry of beta-sheet structures.

The transition moments for the amide bands from beta-sheet peptide structures generally do not exhibit axial symmetry about the director in linearly polarized Fourier transform infrared (FTIR) measurements on oriented systems. The angular dependences of the dichroic ratios of the amide bands are derived for beta-sheet structures in attenuated total reflection (ATR) and polarized transmission experiments on samples that are oriented with respect to the normal to the substrate and are randomly distributed with respect to the azimuthal angle in the plane of the orienting substrate. The orientational distributions of both the beta-strands and the beta-sheets are considered, and explicit expressions are given for the dichroic ratios of the amide I and amide II bands. The dichroic ratio of the amide II band, which is parallel polarized, can yield the orientation of the beta-strands directly, but to specify the orientations of the beta-sheets completely requires measurement of the dichroic ratios of both the amide I and amide II bands, or generally two bands with parallel and perpendicular polarizations. A random distribution in tilt of the planes of the beta-sheets does not give rise to equal dichroic ratios for bands with perpendicular and parallel polarizations, such as the amide I and amide II bands. The results are applied to previous ATR and polarized transmission FTIR measurements on a potassium channel-associated peptide, the Escherichia coli outer membrane protein OmpA, and the E. coli OmpF porin protein in oriented membranes.

Amides↗
PubMed1997

Pneumonia perceptions and management: an ethnographic study in urban squatter settlements of Karachi, Pakistan.

Childhood pneumonia continues to be the second highest contributor to childhood morbidity and mortality in all ethnic groups in Pakistan. Information on community perceptions and management is largely limited to the Punjabi populace. In this study, ethno-specific illness terminologies, recognition and severity indicators and resort to treatment options for childhood pneumonia are explored among the two main ethnic groups in Sindh. Results are based on focus group discussions with 90 caretakers and 16 case history interviews. The findings indicate that pneumonia recognition is almost universal. The main recognition and severity indicator was pasli chalna (chest indrawing) followed by signs and symptoms relating to the quality of breathing and presence of high fever, lethargy and anorexia. Recognition of rapid breathing was low and mostly associated with fever. Exposure to thand (cold) through a variety of mechanisms was perceived to be the dominant causal model. The concept of contagion was virtually non-existent. Despite this, belief in efficacy of allopathic care was very high. Most caretakers reported seeking outside care within one to three days of the onset of symptoms. However, unrealistic expectations of cure often led to change in physicians and treatment regimen, if no improvement was observed by the second day. On the other hand, the quality of care provided by the physicians (both licensed and unlicensed) left much to be desired. Female autonomy and mobility did not appear to be a major constraint in seeking outside care other than for hospitalisation. Implications of these findings for the national acute respiratory infections control programme and future research are discussed

Anthropology, Cultural↗
PubMed1997

Cloning and IgE binding of a recombinant allergen from the mite Blomia tropicalis, homologous with fatty acid-binding proteins.

To characterize the allergens of Blomia tropicalis, a cDNA library was constructed and screened with allergic sera from asthmatic patients. One clone, Bt6, was subcloned and sequenced. The nucleotide sequence of 934 bp length shows a 390-bp reading frame which encodes a 130-amino acid protein with a MW 14.8 kD. No potential glycosylation site was found in the predicted protein. The inferred amino acid sequence has no homology to known allergens. It has a cytosolic fatty acid-binding protein (FABP) signature at 5-22 amino acid residues, 42.3% identity with the Sm14-FABP of Schistosoma mansoni and 36% identity with FABPs from rat, mouse, bovine and human. The protein was expressed as a GST fusion protein and the purified GST-Bt6 used for dot blot, RAST and RAST inhibition assays. The frequency of IgE binding of allergic sera to Bt6 was low (11%) and usually weak. One positive serum did, however, show strong reactivity by RAST and dot blot and Bt6 could inhibit 60% of the IgE binding of this serum to the B. tropicalis extract. These data show that Bt6 encodes a mite FABP with allergenic properties, which are pronounced in some atopic subjects.

Allergens↗