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D Marsh

Publications and source records attributed to D Marsh.

At least 163 records · Page 9Linked to original sources
PubMed1993

Maleimide, iodoacetamide, indanedione, and chloromercuric spin label reagents with derivatized nitroxide rings as ESR reporter groups for protein conformation and dynamics.

The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.

Animals↗
PubMed1993

Demonstration of TGF-beta 1 mRNA by in situ hybridization in normal human fracture healing.

The role of transforming growth factor beta (TGF-beta) in fracture healing has previously been investigated in a rodent model, but not in human material. We investigated TGF-beta 1 gene expression in specimens of callus from normally healing human fractures, using in situ hybridization to a cDNA TGF-beta 1 probe and an autoradiographic disclosure system. TGF-beta 1 mRNA was present in areas of proliferation of mesenchymal tissue, bone, and cartilage. Levels of expression were lower in cells in the fracture hematoma and in differentiated (hypertrophic) chondrocytes. These results are compatible with those found in various animal models using immunohistochemistry and support the view that locally produced TGF-beta 1 is a regulator of fracture repair in humans from the early (mesenchymal proliferation) stage to the stage of remodeling of woven bone. They also indicate that, for TGF-beta 1, animal models accurately reflect human bone repair.

Adolescent↗
PubMed1993

Gangliosides in acetylcholine receptor-rich membranes from Torpedo marmorata and Discopyge tschudii.

The ganglioside composition of membranes enriched in nicotinic acetylcholine receptor (AChR) from the electric rays Discopyge tschudii and Torpedo marmorata has been determined, and compared to that of total electric organ. A ganglioside having the chromatographic mobility of GM2 constitutes the major ganglioside (approximately 60%) in total D. tschudii electric organ, followed by a component with the mobility of GD3 (approximately 10%), and a component running just below GD1a (about 12%). Minor constituents running as GM3 (2%) and as polysialogangliosides (comprising 8-15%) were also observed. Purified native membranes of D. tschudii and T. marmorata displayed a similar profile, except that they were richer in a GM1-like component, and the proportion of GM2-like gangliosides was lower than that in total electric organ. Using a 125I-cholera toxin overlay assay on neuraminidase-treated high-performance thin layer chromatograms, the presence of GM1, GD1a and trace amounts of GD1b and GT1 (or GQ) were detected in D. Tschudii total membranes. Immunocytochemical trechniques showed the co-localization of gangliosides GQ1c/GT1c/GP1c, recognized by the monoclonal antibody Q211, and the AChR at the ventral, innervated face of the electrocyte.

Animals↗
PubMed1993

Insulinlike growth factor gene expression in human fracture callus.

The effects of insulinlike growth factors on bone and cartilage-derived cells in culture have been extensively investigated, but there is little information on their role in vivo in bone, especially in fracture healing. This study investigated insulinlike growth factor (IGF) I and II mRNA expression in normally healing human fractures by in situ hybridization. Endothelial and mesenchymal cells at the granulation tissue stage expressed IGF-II mRNA. At the stage of bone and cartilage formation, osteoblasts and non-hypertrophic chondrocytes expressed mRNA for both IGF-I and II. Some osteoclasts were positive for IGF-II mRNA at the stage of bone remodeling. The greater time span of IGF-II expression relative to IGF-I reflects the predominance of IGF-II in human bone matrix. Taken together with the known effects of IGFs on bone and cartilage cells in vitro, these findings support a role for IGFs in local cellular regulation in human fracture healing.

Adolescent↗
PubMed1993

Investigation of secondary and tertiary structural changes of cytochrome c in complexes with anionic lipids using amide hydrogen exchange measurements: an FTIR study.

The structure of cytochrome c bound to anionic lipid membranes composed of dimyristoyl, dipalmitoyl, or dioleoyl phosphatidylglycerols, or of bovine heart cardiolipin, has been investigated by Fourier transform infrared spectroscopy. Only small changes in secondary structure, as registered by the amide I band of cytochrome c, were observed upon binding at temperatures below that of denaturation of the protein, and these were not coupled to the thermotropic phase transitions of the lipid. The denaturation temperature of the protein decreased by approximately 25-30 degrees upon binding, in a progression which correlated with that of the lipid phase transition temperatures, being approximately 7 degrees lower for complexes with dioleoyl than with dipalmitoyl phosphatidylglycerol. Large changes in the amide proton exchange characteristics, as monitored by the spectral shifts in the amide I band of the protein in D2O, were observed on binding cytochrome c to the lipid membranes. For the slowly exchanging population, the amide deuteration rates of the free protein were nearly independent of temperature, whereas those of the bound protein increased by up to two orders of magnitude over the temperature range from 10 to 40 degrees C. In addition, the extent of exchange differed between the bound and unbound protein. A structural transition in the bound protein was detected as a discontinuous step in Arrhenius plots of the deuterium exchange rates which occurred at a temperature in the region of 22 to 29 degrees C, depending on the lipid, far below that of denaturation. The temperature of this transition was determined by the physical state of the lipid, being 7 degrees lower for the lipids in the fluid state than for those in the gel state, and, for complexes with dimyristoyl phosphatidylglycerol, occurred at an intermediate temperature, being controlled by the lipid chain-melting transition at 27-28 degrees C. These results provide evidence for a coupling of the tertiary structure of the membrane-bound protein with the physical state of the membrane lipids.

Animals↗
PubMed1993

Exchange rates at the lipid-protein interface of the myelin proteolipid protein determined by saturation transfer electron spin resonance and continuous wave saturation studies.

The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.

Animals↗
PubMed1993

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.

Animals↗
PubMed1993

Cause-specific child mortality in a mountainous community in Pakistan by verbal autopsy.

In Pakistan's rural areas population-based cause of death data from systematic verbal autopsies are rare. Using verbal autopsy algorithms with multiple coding and decision rules, we assigned causes of death among 79% of children under age five years dying between July, 1988 and December, 1991 in Oshikhandass, a remote mountainous community in Pakistan's Northern Areas. Main causes of death were pneumonia (44%), diarrhoea (35%), and neonatal sepsis (6%). Combined (main plus associated) analysis revealed 48% died with diarrhoea, 46% with malnutrition, 44% with pneumonia, 15% with neonatal sepsis, and 15% with low birth weight. Median age of death with pneumonia was 2 months, with diarrhoea 8 months. Half died by month 4. The inquiry was well received by villagers. Population-based verbal autopsy surveillance is a cost-effective strategy to guide health managers. Plans are underway to institute it for the surrounding population of 400,000. Creative ways to access, treat and reduce risk among young infants are needed.

Autopsy↗
PubMed1992

Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives.

Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.

Animals↗
PubMed1992

Conventional and saturation transfer EPR spectroscopy of Na+/K(+)-ATPase modified with different maleimide-nitroxide derivatives.

The membranous Na+/K(+)-ATPase from Squalus acanthias has been covalently modified on either Class I or Class II sulphydryl groups using derivatives of 3-(maleimidomethyl)-1-oxyl-2,2,5,5-tetramethylpyrrolidine with substituents of different charge and hydrophobicity attached at the remaining unsubstituted position of the pyrrolidine ring. The substituent groups used were a methyl and a hexyl ester, and di- and tri-methylammonium ethyl esters, as well as the parent underivatized compound. Additionally, another series of maleimide-nitroxides differing (by zero to seven intervening atoms) in the length of the linking group between the maleimide and the pyrrolidine moieties was used. The sites of attachment have been characterized in terms of the rotational mobility and environmental polarity by using conventional and saturation transfer EPR spectroscopy of these spin-labelled reagents. This provides a further sub-classification of the primary Class I and Class II SH-groups on the alpha-subunit of the enzyme, which differ both in their reactivity and influence on the Na+/K(+)-ATPase activity.

Animals↗
PubMed1992

Protein rotational diffusion and lipid/protein interactions in recombinants of bovine rhodopsin with saturated diacylphosphatidylcholines of different chain lengths studied by conventional and saturation-transfer electron spin resonance.

Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7 microseconds), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.

Animals↗
PubMed1992

Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure.

Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗
PubMed1992

The application of pH-sensitive spin labels to studies of surface potential and polarity of phospholipid membranes and proteins.

The effects of pH titration on the EPR spectra of imidazolidine nitroxides located at the surface of mixed bilayers composed of dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC), and at the surface of the protein, human serum albumin (HSA), have been investigated. It is found that the shift in pKa of the amino group of the imidazolidine radical from its value of 4.6 in water depends both on the interfacial polarity (delta pKapol) and on the electrostatic surface potential (delta pKael) when it is positioned at the bilayer/water interface by an anchoring hydrocarbon tail. The polarity shift is determined to be: delta pKapol = -1.3 units at the surface of DMPC bilayers at 17 degrees C, corresponding to an effective interfacial dielectric constant of epsilon approximately 37, and depends on the temperature with a coefficient of d delta pKapol/dT approximately -0.01 per degree. The electrostatic shift at the surface of DMPG bilayers is delta pKael = +1.6 units in 0.1 M KCl, which corresponds to an electrostatic surface potential of -95 mV. This electrostatic shift depends strongly both on ionic strength and on the fraction of charged lipid in the DMPC/DMPG mixtures, in a manner that agrees with the predictions of electrostatic double-layer theory. It is found that the shift in pKa of an imidazolidine radical covalently bound at the surface of HSA is determined mainly by the surface electrostatics (delta pKapol approximately 0) and corresponds to an electrostatic potential of +33 mV in 0.01 M KCl at a pH below the isoelectric point of the protein.

Dimyristoylphosphatidylcholine↗
PubMed1992

Interfacial ionization and partitioning of membrane-bound local anesthetics.

Consideration of the interfacial protonation equilibria of membrane-associated amphiphiles indicates that the partition coefficients of the protonated and unprotonated species will differ considerably. The partition coefficients of the charged and uncharged forms of spin-labelled myristic acid in dimyristoylphosphatidylcholine bilayer dispersions have been measured by EPR spectroscopy and found to be approximately 140-fold higher for the protonated acid than for the dissociated salt form. This ratio of partition coefficients is found to be in good agreement with that predicted from the interfacial shift in pKa of the fatty acid on its partitioning into the membrane. The latter was determined from the changes in the EPR spectra of the membrane-associated fatty acid with pH and was found to be +2.1 pH units. The interfacial shifts in pKa for a series of spin-labelled analogues of tertiary amine local anaesthetics have been determined from the pH dependence of the partition coefficients in dimyristoylphosphatidylcholine bilayer dispersions and are found mostly to be in the range of approx. -1.0 to -1.5 pH units, corresponding to a 10- to 30-fold higher partition coefficient of the uncharged base compared with that of the charged ammonium form.

Anesthetics↗
PubMed1992

Determination of fluid and gel domain sizes in two-component, two-phase lipid bilayers. An electron spin resonance spin label study.

The average sizes of fluid and gel domains in the two-component, two-phase system formed from mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine were determined from an analysis of the electron spin resonance spectral lineshapes of a dimyristoyl phosphatidylcholine-nitroxide spin label as a function of spin label concentration. The ratio, R, of the intensities measured at two magnetic field strengths was found to be diagnostic of a statistical distribution of spin labels in disconnected domains. R is defined as V'/2Vpp, where Vpp is the maximum intensity and V' is the intensity at a position in the wings of a first derivative electron spin resonance line that is a constant multiple of the peak-to-peak linewidth. The intensity ratio for Gaussian or Voigt lineshapes is less than or equal to the value for a Lorentzian lineshape. The intensity ratio was found to be greater than the value for a Lorentzian line when spectra from disconnected domains containing a statistical distribution of spin labels undergoing spin-spin interactions were summed. The intensity ratio, R, calculated by spectral simulations as a function of the average number of labels per domain, N, was found to increase to a maximum with increasing N and then to decrease. The dependence on spin label concentration of the experimentally measured intensity ratios paralleled this predicted behavior. A method is presented to calculate the average number of lipids per fluid or gel domain based on a knowledge of R, and of the distribution of the spin label between the fluid and gel phases determined from the phase diagram. The results demonstrate that the number of lipids per domain increases linearly from a fixed number of nucleation sites, as the fraction of the phase that is disconnected increases. At any given mole fraction of the particular phase, the gel domains are bigger than the fluid domains because they have a lower nucleation density. The results also suggest that the disconnected domains are, in most cases, nonrandomly distributed in the plane of the bilayer.

Dimyristoylphosphatidylcholine↗