Search PubMed⌕ Search

Biomedical subjects

A Watts

Publications and source records attributed to A Watts.

At least 127 records · Page 7Linked to original sources
PubMed1993

The effect of general anesthetics on the dynamics of phosphatidylcholine-acetylcholine receptor interactions in reconstituted vesicles.

The interaction of general anesthetics at the lipid/protein interface of the nicotinic acetylcholine receptor reconstituted in dioleoylphosphatidylcholine bilayers at various lipid/protein ratios has been studied using the electron spin resonance spectra of phosphatidylcholine spin-labeled at the fourteenth acyl carbon (14-PCSL). In addition to the bilayer spectrum, the spin label reported a more motionally restricted environment whose contribution increased with increasing protein/lipid ratio. Exchange between these two environments occurred at a rate of approx. 6 x 10(7) s-1. The motionally restricted, protein-associated 14-PCSL had a rotational correlation time of about 10-20 ns, an order of magnitude slower than when in the bilayer. Addition of 1-hexanol (up to 16 mM) to the reconstituted receptor perturbed the acyl chains of the bulk lipid phase, but the motional properties of the lipid acyl chains at the protein/lipid interface near the membrane center were not significantly perturbed on the EPR motional time-scale. Similarly, anesthetics that were less effective at perturbing the bilayer, such as pentobarbital (up to 2 mM) and isoflurane (7 mM), did not perturb the lipid/protein interface on the conventional EPR motional time scale.

Anesthesia, General↗
PubMed1993

2H NMR lineshapes of immobilized uniaxially oriented membrane proteins.

As a method for the structure determination of integral membrane proteins or other large macromolecular complexes, a solid state 2H NMR approach is presented, capable of measuring the orientations of individual chemical bond vectors. In an immobilized uniaxially oriented sample, the bond angle of a deuterium-labelled methyl group relative to the axis of ordering can be calculated from the quadrupole splitting in the "zero-tilt" spectrum where the sample normal is aligned parallel to the spectrometer field direction. However, since positive and negative values of this splitting cannot be distinguished, there may appear to be two solutions, of which only one describes the correct molecular geometry. We show that it is possible to determine the bond angle uniquely between 0 degree and 90 degrees, by analysing the lineshapes of a tilt series of spectra acquired over different sample inclinations. The lineshape equation describing such oriented 2H NMR spectra will be derived (for asymmetry parameter eta = 0) and discussed, with an illustration of the various linebroadening effects from which the orientational distribution function in the macroscopically ordered system can be determined. This strategy is then applied to specifically deuterium-labelled retinal in dark-adapted bacteriorhodopsin, prepared in a uniaxially oriented sample from purple membrane fragments. From the quadrupole splitting in the zero-tilt spectrum and by lineshape simulations, the deuteromethyl group at C20 on retinal is found to make an angle of 32 degrees +/- 1 degree with the membrane normal, and the sample mosaic spread to be around +/- 8 degrees. The resulting orientation of retinal is in excellent agreement with its known structure in bacteriorhodopsin, and together with the results on other methyl groups it will be possible to construct a detailed picture of the chromophore in the protein binding pocket.

Bacteriorhodopsins↗
PubMed1993

Dynamics in a protein-lipid complex: nuclear magnetic resonance measurements on the headgroup of cardiolipin when bound to cytochrome c.

Deuterium and phosphorus nuclear magnetic resonance (NMR) has been used to investigate the dynamics of slow motional processes induced in bilayer cardiolipin upon binding with cytochrome c. 31P NMR line shapes suggest that protein binding induces less restricted, isotropic-like motions in the lipid phosphates within the ms time scale of this measurement. However, these motions impart rapid transverse relaxation to methylene deuterons adjacent to the phosphate in the lipid headgroup and so did not feature strongly in the NMR line shapes recorded from these nuclei by using the quadrupolar echo. Nonetheless, motional characteristics of the headgroup deuterons were accessible to a dynamic NMR approach using the Carr-Purcell-Meiboom-Gill multiple-pulse experiment. Compared to the well-studied case of deuterons in fatty acyl chains of bilayer phosphatidylcholine, the motions determining the 2H spin transverse relaxation in the headgroup of bilayer cardiolipin were much faster, having a lower limit in the 5-10 kHz range. On binding with cytochrome c, the T2 effecting motions in the cardiolipin headgroup became faster still, with rates comparable to the residual quadrupolar coupling frequency of the headgroup deuterons (approximately 25 kHz) and so coincided with the time scale for recording the quadrupolar echo (approximately 40 microseconds). It is concluded that the headgroup of cardiolipin does not exclusively report localized dynamic information but is particularly sensitive to collective motions occurring throughout the bilayer molecules. Although the rates of collective modes of motion may be dependent on the lipid type in pure lipid bilayers, these low-frequency fluctuations appear to occupy a similar dynamic range in a variety of lipid-protein systems, including the natural membranes.

Animals↗
PubMed1993

Development and harmonisation of the norms and parameters in biological testing protocols designed for root canal sealing materials within the European Community.

The EC has mounted the BIOMED 1 as a specific programme to facilitate cooperative research programmes in identified areas of interest throughout Europe. They have recently announced support under this programme for a joint initiative designed to evaluate in vitro and in vivo methods of biological testing of root canal sealing materials. This proposal was prepared by a group of research workers in the field. It provides a summary of the current suggested protocols for the biological testing of dental materials and then provides a detailed protocol of the studies to be carried out in the joint programme which commenced in January 1993. The authors are publishing the proposal to invite comments from colleagues and interested parties.

Animals↗
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↗