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Biomedical subjects

E Oldfield

Publications and source records attributed to E Oldfield.

At least 91 records · Page 5Linked to original sources
PubMed1982

Endocarditis caused by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a small, gram-negative bacillus with fastidious growth requirements, is a rare cause of endocarditis. The clinical course of this disease is characterized by long duration of symptoms before diagnosis (average of three months), prolonged incubation before growth (average of six days), and a high embolic rate. The optimal antimicrobial regimen cannot be determined from available data, but the combination of ampicillin and an aminoglycoside may offer the best chance for cure. Two cases are presented, one successfully treated. Previously reported cases are reviewed.

Actinobacillus Infections↗
PubMed1981

X-ray diffraction and calorimetric study of anhydrous and hydrated N-palmitoylgalactosylsphingosine (cerebroside).

Differential scanning calorimetry and X-ray diffraction of anhydrous and hydrated N-palmitoylgalactosylsphingosine (NPGS) show evidence of complex polymorphic behavior and interconversions between stable and metastable structural forms. Anhydrous NPGS exhibits three lamellar crystal forms (A, B, and B') at temperatures below 143 degrees C and a liquid-crystal form between 143 and 180 degrees C before melting to an isotropic liquid at 180 degrees C. The crystal B leads to liquid-crystal transition is accompanied by an enthalpy change, delta H, of 11.2 kcal/mol of NPGS, while a relatively small enthalpy change (delta H = 0.8 kcal/mol) marks the liquid-crystal leads to liquid transition. The A and B' crystal forms do not hydrate readily at room temperature. When heated, crystal form A in the presence of water undergoes an exothermic transition at 52 degrees C to produce a thermodynamically stable hydrated crystal E form. X-ray diffraction shows that this stable bilayer crystal form has a highly ordered hydrocarbon chain packing arrangement; melting to the bilayer liquid-crystal form occurs at 82 degrees C with a large enthalpy change, delta H = 17.5 kcal/mol of NPGS. A complex liquid-crystal leads to crystal transition is observed on cooling; the cooling rate independent exotherm involves the transition of the hydrated liquid crystal to an intermediate metastable crystal form identical with anhydrous crystal form A. The subsequent cooling rate dependent step involves the conversion of the metastable crystal form A to the stable crystal form E. We suggest that hydrated crystal form E is stabilized by both a highly ordered chain packing mode and a lateral intermolecular hydrogen bonding network involving the sphingosine backbone, the galactosyl group, and interbilayer water molecules. Although disruption of both the specific hydrogen chain packing and H-bonding networks occurs at the high enthalpy transition to the bilayer liquid-crystal L alpha form, these two types of interactions are not reestablished simultaneously on cooling. First, recrystallization of the hydrocarbon chain accompanies removal of water from the lipid interface, leading to "dehydrated" metastable crystal form A. This is followed by a time-dependent, temperature-dependent hydration process which allows a rearrangement of the hydrogen-bonding matrix. Alterations in the NPGS-NPGS and NPGS-water interactions accompany further changes in the hydrocarbon chain packing and lead to the formation of the stable E form.

Animals↗
PubMed1981

Dynamics of amino acid side chains in membrane proteins by high field solid state deuterium nuclear magnetic resonance spectroscopy. Phenylalanine, tyrosine, and tryptophan.

We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring "flipping."

Bacteriorhodopsins↗
PubMed1981

First observation of amino acid side chain dynamics in membrane proteins using high field deuterium nuclear magnetic resonance spectroscopy.

We have obtained the first deuterium NMR spectra of an individual membrane protein, bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. Biosynthetic isotopic enrichment with [gamma-2H6]valine and high field Fourier transform operation permitted rapid data acquisition on intact membranes, including measurement of relaxation times. At some temperatures high quality spectra could be obtained in less than 1 s. [U-14C]Valine tracer studies indicate that less than or equal to 2% of valine added to the growth medium is broken down and incorporated into other membrane constituents. The NMR results indicate that the valine side chain is a rather rigid structure. Motion about C alpha-C beta is slow (less than 10(5) s-1) at growth temperature, While motion about C beta-C gamma is as expected fast (much greater than 10(5) s-1) at all accessible temperatures. The activation energy for methyl group rotation from spin-lattice relaxation data between -75 and 53 degrees C is approximately 2.4 kcal/mol, in good agreement with previous 1H NMR studies on solid alkanes. Preliminary data on [gamma-2H6]valine-labeled Acholeplasma laidlawii B (PG9) cell membranes are also presented. Our results strongly suggest that it should now be possible to observe in great detail the motions of any type of amino acid side chain in membrane proteins, including the effects of lipid composition on protein dynamics.

Amino Acids↗
PubMed1981

Nuclear magnetic resonance of heme protein crystals. General aspects.

A new technique capable of determining the static and dynamic structures of heme protein crystals is reported. It is shown that microcrystals of a variety of paramagnetic heme proteins, suspended in approximately 90% saturated (NH4)2SO4, may be perfectly aligned by an intense static external magnetic field, H0, due to the large anisotropy in the magnetic susceptibility of the protein caused by the paramagnetic center. Myoglobin from sperm whale (Physeter catodon) was isotopically enriched at the C epsilon methyl groups of methionine residues 55 and 131 with either 13C or 2H and studied in the crystalline solid state by 2H-quadrupole echo and 13C-Fourier transform nuclear magnetic resonance spectroscopy. It was found that suspensions of both high (S = 5/2) and low (S = 1/2) spin ferric forms of the labeled protein were ordered, the axis of ordering being approximately perpendicular to the low temperature minimum g tensor valve, even though upper Kramers levels are populated at room temperature. The paramagnetic CoII derivative "coboglobin" showed similar ordering behavior, but the diamagnetic carboxymyoglobin was unaffected. The magnetic ordering method permits the recording of "single crystal" NMR spectra from microcrystalline arrays of proteins which cannot be prepared in large enough form (approximately 1 cm3) for single crystal NMR spectroscopy and thereby allows the resolution and assignment of numerous single atom sites in the crystalline solid state. The information from a "single crystal" NMR spectrum combined with that obtained on the crystal powder allows for the direct determination of (i) the spatial orientation of the particular labeled residue within the protein crystal and (ii) the rates and types of side chain motion. Resonances were assigned by spin label broadening experiments and by use of existing x-ray data to predict 2H-NMR spectra. This new technique opens up the possibility of determining directly the dynamic structure of protein crystals and of comparing the structures of proteins in the crystalline solid state with that in solution and is applicable to other heme proteins, e.g. catalase.

Animals↗
PubMed1980

Deuterium NMR of specifically deuterated fluorine spin probes.

Deuterium nuclear magnetic resonance spectra (at 55.3 MHz) have been obtained of 19F-2H double-labeled phospholipids in pure lipid bilayers, and of 2H-labeled lipid in a 19F-labeled bilayer, as a function of concentration, to assess the perturbing influence of 19F sites in lipid hydrocarbon chains. Order parameters of 2H-labeled sites adjacent to C-8 myristic fluorine probes in pure lipid bilayers, and 19F spin label order parameters themselves, are about 30% lower than those deduced from the use of nonperturbing 2H probes. The effect is intramolecular rather than intermolecular and presumably represents increased gauche states due to the increased size of the 19F label. This effect is consistent with the view that difluoromethylene fatty acyl chains function in a manner approximating that of unsaturated fatty acyl chains. The differences disappear in the presence of cholesterol at very high order parameters (Smol approximately 0.8 to 0.9). These results represent the first attempt at elucidating the perturbing effects of a high sensitivity probe (19F) and indicate that caution must be used when using spectroscopic probes to deduce the absolute magnitude of hydrocarbon chain order parameters.

Deuterium↗
PubMed1980

Laser Raman spectroscopic study of specifically deuterated phospholipid bilayers.

Raman spectra of 1,2-dimyristoyl-sn-glycero-3-phosphocholines specifically deuterated in the 2 chain at one of positions 3, 4, 6, 10, 12, and 14 have been obtained as a function of temperature. The frequencies of the CD2 vibrational stretching modes depend on the position of the labeled CD2 group, being maximum at position 3 of the acyl chain and then decreasing until they become constant beyond position 6. This frequency dependence is interpreted in terms of the inductive effect of the charge distribution of the acyl chain carboxyl group. In both gel and liquid-crystal phases, the Raman line widths depend on the position of the CD2 group, being minimum at position 6 and increasing toward both ends of the hydrocarbon chain. The width of the CD stretching bands abruptly increases at the phase transition temperature. The magnitude of the increase depends upon the position of the label, increasing almost linearly up to position 10 and then decreasing for positions 12 and 14. The spectra for the CD2 group at position at position 3 and the terminal CD3 group are almost the same in both phases. These results are interpreted in terms of the effects of hydrocarbon chain organization on the vibrational modes.

Deuterium↗
PubMed1979

Physical studies of cell surface and cell membrane structure. Determination of phospholipid head group organization by deuterium and phosphorus nuclear magnetic resonance spectroscopy.

Phospholipid head group conformations in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), DPPC-cholesterol, and DPPE-cholesterol dispersions, in excess water above the pure lipid gel to liquid-crystal phase transition temperature, have been calculated by using comparisons between experimental 2H and 31 P NMR spectral parameters and theoretical results obtained from a plausible model of head group motions. The new calculations are compared with results obtained in previous studies [Seelig, J., Gally, H. U., & Wohlgemuth, R. (1977) Biochem, Biophys. Acta 467, 109--117; Brown, M. F. & Seelig, J. (1978) Biochemistry 17, 381--384; Seelig, J., & Gally, H. U. (1976) Biochemistry 15, 5199--5204] and are shown to agree qualitatively under certain highly restrictive conditions. Under more general conditions, it is shown that many possible solutions are generated but that these may often be separated into a small number of likely conformations in which the head group torsion angles are restricted to specific ranges rather than to a discrete set of values. There is no NMR evidence, however, to support the notion that there are only single conformational solutions to the NMR measurements for the above phospholipid systems.

Cell Membrane↗
PubMed1979

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.

Animals↗
PubMed1979

Physical studies of cell surface and cell membrane structure. Deuterium nuclear magnetic resonance investigation of deuterium-labelled N-hexadeconoylgalactosylceramides (cerebrosides).

1. Deuterium Fourier transform nuclear magnetic resonance spectra of a series of N-palmitoylgalactosylceramides (cerebrosides) specifically labelled with deuterium at one of positions 2', 6', 10' and 16' of the acyl chain, or in the C-6 hydroxymethyl group of the galactose residue, have been obtained using a spin-echo technique at 34.1 MHz with a homebuilt superconducting magnet spectrometer. 2. The effects of temperature and cholesterol on the deuterium spectra have been investigated. The results indicate, when compared at the same reduced temperature, that the hydrocarbon chain organization in the liquid crystalline phase of palmitoylgalactosylceramide is essentially identical to that seen in similar chain length glycerophospholipids. In particular, two sets of quadrupole splittings are seen for a 2'-labelled N-palmitoylgalactosylceramide, indicating non-equivalent deuterons as noted previously for phospholipids. 3. Two sets of quadrupole splittings are observed for the headgroup C-6-labelled N-palmitoylgalactosylceramide. It is proposed that these signals arise from the enantiomeric R and S lipids, and that motion of the hydroxymethyl group is slow (greater than 10(-5) S). These results suggest the presence of a hydrogen bond network in the polar headgroup region. 4. The effects of cholesterol on the deuterium spectra of N-palmitoylgalactosylceramide-labelled as C2H3 in the terminal methyl group, at 1:1 mol ratios and in excess water below the crystal to liquid-crystal phase transition temperature (Tc) of the pure lipid (82 degrees C), are different to the effects seen with the phosphatidylcholine-cholesterol system. The spectra below Tc are characterised by two overlapping powder patterns, one with a quadrupole splitting of approx. 6 kHz (fluid liquid-crystalline phase) and one with a quadrupole splitting of about 20--25 kHz (crystal or gel-state lipid). Exchange between these two environments is therefore slow, leading to the possibility of characterising the cerebroside-cholesterol phase diagram using deuterium nuclear magnetic resonance spectroscopy.

Cell Membrane↗