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Biomedical subjects

A Watts

Publications and source records attributed to A Watts.

At least 19 recordsLinked to original sources
PubMed1992

Structure determination of the cyclohexene ring of retinal in bacteriorhodopsin by solid-state deuterium NMR.

The orientation and conformation of retinal within bacteriorhodopsin of the purple membrane of Halobacterium halobium was established by solid-state deuterium NMR spectroscopy, through the determination of individual chemical bond vectors. The chromophore ([2,4,4,16,16,17,17,17,18,18-2H11]retinal) was specifically deuterium-labeled on the cyclohexene ring and incorporated into the protein. A uniaxially oriented sample of purple membrane patches was prepared and measured at a series of inclinations relative to the spectrometer field. 31P NMR was used to characterize the mosaic spread of the oriented sample, and computer simulations were applied in the analysis of the 2H NMR and 31P NMR spectral line shapes. From the deuterium quadrupole splittings, the specific orientations of the three labeled methyl groups on the cyclohexene ring could be calculated. The two adjacent methyl groups (on C1) of the retinal were found to lie approximately horizontal in the membrane and make respective angles of 94 degrees +/- 2 degrees and 75 degrees +/- 2 degrees with the membrane normal. The third group (on C5) points toward the cytoplasmic side with an angle of 46 degrees +/- 3 degrees. These intramolecular constraints indicate that the cyclohexene ring lies approximately perpendicular to the membrane surface and that it has a (6S)-trans conformation. From the estimated angle of the tilt of the chomophore long axis, it is concluded that the polyene chain is slightly curved downward to the extracellular side of the membrane.

Bacteriorhodopsins↗
PubMed1992

Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study.

The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Isotopes↗
PubMed1992

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.

Bacteriorhodopsins↗
PubMed1992

Effect of bacteriorhodopsin on the orientation of the headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in bilayers: a 31P- and 2H-NMR study.

Bacteriorhodopsin (BR), purified from the halophilic bacterium, Halobacterium halobium, has been separated from the endogenous purple membrane lipids and reconstituted by detergent dialysis into bilayers of the zwitterionic phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), which was selectively deuterated at the headgroup in the choline alpha- and beta-methylene segments and the choline gamma-methyl groups. Complexes of DMPC/BR contents from 67:1 to 222:1 (mol/mol) were produced under conditions to promote formation of large vesicles (mean diameters 600-700 nm). The magnitudes of the 2H-NMR quadrupole splittings recorded from the deuterium-labelled headgroup segments, and the 31P-NMR chemical shift anisotropy (CSA) of the phosphate group appeared to vary linearly with the BR content in the complexes over the range of DMPC/BR ratios studied. On increasing the proportion of BR in the DMPC-BR complexes, the 2H-NMR quadrupole splittings measured from the choline gamma-methyl groups and the beta-methylene segments and the 31P-NMR CSA increased in magnitude, while the 2H-NMR quadrupole splitting from the alpha-methylene segment decreased. Such opposing changes in the choline alpha- and beta-methylene segment quadrupole splittings are similar to those reported on increasing the proportion of positively charged amphiphile at the bilayer surface (Seelig et al. (1987) Biochemistry 26, 7535-7541). It is suggested that BR presents a net positive charge to the phosphocholine headgroups at the protein/lipid interface.

Bacteriorhodopsins↗
PubMed1992

Structural stability of the erythrocyte anion transporter, band 3, in native membranes and in detergent micelles.

The exothermic thermal denaturation transition of band 3, the anion transporter of the human erythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190-300 kcal mol-1) with an accompanying decrease in thermal denaturation temperatures (48-60 degrees C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75 degrees C) and DIDS-insensitive (62 degrees C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant differences was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C12E8) are required for optimal thermal stability of band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Saturation transfer, continuous wave saturation, and saturation recovery electron spin resonance studies of chain-spin labeled phosphatidylcholines in the low temperature phases of dipalmitoyl phosphatidylcholine bilayers. Effects of rotational dynamics and spin-spin interactions.

The saturation transfer electron spin resonance (STESR) spectra of 10 different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain have been investigated in the low temperature phases of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The results of continuous wave saturation and of saturation recovery measurements on the conventional ESR spectra were used to define the saturation properties necessary for interpreting the STESR results in terms of the chain dynamics. Spin labels with the nitroxide group located in the center of the chain tended to segregate preferentially from the DPPC host lipids in the more ordered phases, causing spin-spin interactions which produced spectral broadening and had a very pronounced effect on the saturation characteristics of the labels. This was accompanied by a large decrease in the STESR spectral intensities and diagnostic line height ratios relative to those of spin labels that exhibited a higher degree of saturation at the same microwave power. The temperature dependence of the STESR spectra of the different spin label isomers revealed a sharp increase in the rate of rotation about the long axis of the lipid chains at approximately 25 degrees C, correlating with the pretransition of gel phase DPPC bilayers, and a progressive increase in the segmental motion towards the terminal methyl end of the chains in all phases. Prolonged incubation at low temperatures led to an increase in the diagnostic STESR line height ratios in all regions of the spectrum, reflecting the decrease in chain mobility accompanying formation of the subgel phase. Continuous recording of the central diagnostic peak height of the STESR spectra while scanning the temperature revealed a discontinuity at approximately 14-17 degrees C, corresponding to the DPPC subtransition which occurred only on the initial upward temperature scan, in addition to the discontinuity at 29-31 degrees C corresponding to the pretransition which displayed hysteresis on the downward temperature scan.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Pulp responses to two strains of bacteria isolated from human carious dentine (L. plantarum) (NCTC 1406) and S. mutans (NCTC 10919).

A series of studies has been conducted in which monoinfected gnotobiotic rats were used to study the responses of the dental pulp to micro-organisms isolated from carious lesions in dentine. In this study pulp responses to L. plantarum (formerly odontolyticus) (NCTC 1406) in pure culture and in combination with S. mutans (NCTC 10919) are reported. The incidence of inflammation/necrosis/dentine bridge formation observed in animals monoinfected with L. plantarum was similar to that reported in previous germ-free studies. There was a greater incidence of dentine bridge formation in rats monoinfected with L. plantarum compared with those monoinfected with S. mutans. When the two organisms were combined, periapical inflammation was observed in 14% of the teeth examined after 28 days, but there was no significant difference in the incidence of dentine bridge formation. Considerable variation in the density of staining of the two microorganisms in histological sections was observed.

Animals↗
PubMed1992

Pulp response to, and cariogenicity of, a further strain of Streptococcus mutans (NCTC 10832).

The pulpal response to and cariogenicity of a third strain of Streptococcus mutans, namely S. mutans (NCTC 10832), was studied in monoinfected gnotobiotic rats of the Fischer strain using the techniques described previously by the present authors. Unlike S. mutans (NCTC 10449 and 10919), S. mutans (NCTC 10832) was associated with the presence of inflammatory cell infiltrates in the coronal pulp of a small number of teeth and extensive periapical inflammation 28 days after the creation of untreated pulpal exposures. S. mutans (NCTC 10832) was associated with the presence of extensive pulpal necrosis and reduced dentine bridge formation. These changes were similar to those noted with the other two strains of S. mutans. S. mutans (NCTC 10832) was non-cariogenic in monoinfected gnotobiotic rats of the Fischer strain.

Animals↗
PubMed1992

Continuing education for general dental practitioners using a printed distance learning programme.

This paper describes the development of a printed distance learning programme by a coordinated team approach. The programme has two components, a resource book and three patient management problem leaflets or challenges and is designed to update general dental practitioners on the management of fissure caries. It includes strategies to meet the educational needs of three groups of general dental practitioners with different educational requirements. It is concluded that the approach described meets the CRISIS criteria for effective continuing education and offers a useful approach to distance learning in dentistry.

Education, Dental, Continuing↗
PubMed1992

The bony crescent sign--a new sign of facial nerve schwannoma.

Schwannomas of the facial nerve are rare slowly growing lesions that have a predilection for the geniculate ganglion. Radiological evaluation is important in their diagnosis and in the assessment of their extent. In our series of 4 cases the facial nerve schwannoma was seen on high resolution CT as a soft tissue mass bounded anteriorly by a thin rim of bone. This bony crescent sign is a previously undescribed sign of facial nerve schwannoma which appears on the basis of this small series to be strongly indicative of the presence of this tumour. Schwannomas are relatively uncommon intracranial tumours. They most commonly involve the acoustic nerve followed in frequency by the trigeminal nerve. Other cranial nerves are rarely involved. Facial nerve schwannomas occurring within the petrous temporal bone are very rare. Their diagnosis may be missed prospectively even when appropriate CT scans are performed. Even in retrospect the site of abnormality may be difficult to identify, especially if there is an associated middle ear mass such as a cholesteatoma. Lesions occurring in the petrous area are all rare. The differential diagnosis includes cholesterol granuloma, epidermoid, carotid aneurysm and, very rarely, primary and secondary bone tumours. We describe a new sign associated with facial nerve schwannoma on CT, that of a bony crescent. Recognition of this sign makes those tumours arising in the region of the geniculate ganglion easy to diagnose prospectively.

Adolescent↗
PubMed1991

Characterization of phospholipid compositions and physical properties of DMPC/bacteriorhodopsin vesicles produced by a detergent-free method.

Homogeneous complexes of bacteriorhodopsin (BR) from Halobacterium halobium purple membrane (PM) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) have been produced by a detergent-free process in which bovine liver non-specific phospholipid transfer protein (nsTP) promotes net transfer of DMPC from small unilamellar vesicles directly into PM. The number of DMPC molecules incorporated per BR monomer follows a close to linear dependence with the relative proportions of DMPC and PM added to the initial mixture over the ranges studied. The resulting complexes, with total lipid phosphate/BR contents of between 31:1 and 152:1 (mole/mole), were purified free from any remaining unincorporated DMPC by sucrose density gradient centrifugation. Broad line 31P-NMR spectra and partitioning studies with the nitroxide spin label, Tempo, confirm that the BR and DMPC coexist in bilayer complexes. Quantitative analysis of high resolution 31P-NMR spectra from complexes after solubilization in 4% SDS revealed 74-84% of the major PM phospholipid to be retained in the complexes.

Animals↗
PubMed1991

Partitioning behaviour of 1-hexanol into lipid membranes as studied by deuterium NMR spectroscopy.

Deuterium nuclear magnetic resonance (NMR) spectroscopy was used to study the partitioning behaviour of 1-hexanol specifically deuterated in the alpha-position into model lipid bilayers. In all systems studied, the observed deuterium NMR lineshapes were time-dependent. Initially, 1-hexanol-d2 gave rise to an isotropic deuterium resonance with a different chemical shift from that of aqueous 1-hexanol-d2. After equilibration over a period of days, a broader spectral component characteristic of a spherically-averaged powder-pattern was observed. The quadrupole anisotropy of the 1-hexanol-d2 giving rise to the broad spectrum depended upon the cholesterol content of the membrane. From quantitation of the anisotropic to isotropic deuterium NMR spectra, the partition coefficients of 1-hexanol-d2 in a number of bilayer systems (asolectin and phosphatidylcholine bilayers (the latter with and without cholesterol] were determined. The partitioning of 1-hexanol-d2 into red blood cell membranes, and a suspension of lipids extracted from red blood cell membranes, was also examined. It is suggested that 1-hexanol, and probably other lipophiles, can partition to either the bilayer surface or the bilayer interior in a time-dependent manner.

Deuterium↗
PubMed1991

A NMR investigation on the interactions of the alpha-oligomeric form of the M13 coat protein with lipids, which mimic the Escherichia coli inner membrane.

The interaction of the M13 bacteriophage major coat protein in the alpha-oligomeric form with specifically deuterated phospholipid headgroups which mimic the Escherichia coli inner membrane, has been studied using NMR methods. As can be seen from the deuterium NMR spectra obtained with headgroup trimethyl deuterated DOPC, the coat protein in the alpha-oligomeric form does not give rise to trapped lipids as observed with M13 coat protein in the beta-polymeric form (Van Gorkom et al. (1990) Biochemistry 29, 3828-3834). The quadrupolar splittings of the alpha headgroup methylene deuterons of deuterated phosphatidylcholine and phosphatidylethanolamine decrease, whereas the quadrupolar splittings of the beta headgroup methylene deuterons of the two lipids increase with increasing protein content. All deuterated segments in the phosphatidylglycerol headgroup show the same relative decrease of the NMR quadrupolar splittings. These results are interpreted in terms of a change in torsion angles of the methylene groups, induced by positive charges, probably lysine residues of the protein at the membrane surface. For all lipid bilayer compositions studied the head-group perturbations are similar. It is concluded that there is no strong specific interaction between one of the lipid types examined and the M13 coat protein. From the spin-spin (T2e) relaxation time and spin-lattice (T1z) relaxation time of all deuterated lipids it is concluded that at the bilayer surface only slow motions are affected by the M13 coat protein.

Capsid↗
PubMed1991

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1. Evidence from deuterium NMR measurements.

Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare [N epsilon, N epsilon-C2H3]lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give [amide-2H]cytochrome c or more selective labeling of just histidine residues in [epsilon-2H]histidine cytochrome c. Deuterium NMR measurements on [N epsilon, N epsilon-C2H3]lysyl cytochrome c in the solid state showed restricted motions, fairly typical of the behavior of aliphatic side-chain sites in proteins. The [amide-2H]cytochrome c provided "immobile" amide spectra showing that only the most stable backbone sites remained labeled in this derivative. Relaxation measurements on the aqueous solution of [amide-2H]cytochrome c yielded a rotational correlation time of 7.9 ns for the protein, equivalent to a hydrodynamic diameter of 4.0 nm, just 0.6 nm greater than its largest crystallographic dimension. Similar measurements on [epsilon-2H]histidine cytochrome c in solution showed that all labeled histidine residues were also "immobile" compared with the overall reorientational motion of the protein. The interaction with cardiolipin bilayers appeared to create a high degree of mobility for the side-chain sites of [N epsilon, N epsilon-C2H3]lysyl cytochrome c and perturbed backbone structure to instantaneously release all deuterons in [amide-2H]cytochrome c. The [epsilon-2H]histidine cytochrome c derivative, when complexed with cardiolipin, failed to produce any detectable wide-line 2H NMR spectrum, demonstrating that the overall reorientational motion of bound protein was not isotropic on the NMR time scale, i.e., tau c greater than 10(-7)s.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiolipins↗
PubMed1991

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.

31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.

Cardiolipins↗