Cholesterol metabolism: the main pathway acting downstream of cytochrome P450 oxidoreductase in skeletal development of the limb.

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.

A novel approach to the visualisation of heat-induced structural change in bone.

A new approach to visualising heat-induced change in bone was attempted. This was an attempt to counter the serious limitations of existing analytical methods yet still allow for the examination of subtle changes that occur due to burning. A new form of Magnetic Resonance Imaging was deemed to fulfil this remit. Preliminary tests were performed and proved successful in creating clear, well-defined images of progressive heat-induced structural changes in bone. The implications for improving our understanding of heat-induced change, and therefore our methods of human identification, are significant.

Non spiral and spiral (helical) flow patterns in stenoses. In vitro observations using spin and gradient echo magnetic resonance imaging (MRI) and computational fluid dynamic modeling.

AIM: Physiological blood flow patterns are themselves poorly understood despite their impact on arterial disease. Stable spiral (helical) laminar flow (SLF) has been observed in normal subjects. The purpose of the present study is to develop a method of magnetic resonance (MR) flow pattern visualization and to analyze spiral and non-spiral flow patterns with and without luminal narrowing in vitro. The flow conditions were then modeled using computational fluid dynamics (Star-CD). METHODS: Laminar integrity was examined in a flow-rig using spin and gradient echo magnetic resonance imaging (MRI) in non-stenosed and stenosed conduits in the presence of non-spiral and spiral flow. RESULTS: No difference was observed in a non-stenosed conduit between non-spiral and spiral flow. In the presence of a stenosis spiral flow preserves flow velocity coherence whereas non-spiral flow increasingly lost coherence beginning proximal to the stenosis. Computational fluid dynamic modeling of the in vitro experiment showed marked differences between the 2 flow patterns. Non-spiral flow produced greater inwardly directed forces just beyond the stenosis and greater outward pressures at more distal sites. The near wall turbulent energy was up to 700% less with spiral flow over non-spiral flow beyond the stenosis. CONCLUSIONS: Spiral flow appears to offer clear flow profile stabilizing advantages over non-spiral flow, by significantly reducing the turbulence caused by a stenosis. Spiral flow also produces lower forces acting on the vessel wall.

Nuclear magnetic resonance imaging (MRI) of diesel oil migration in estuarine sediment samples.

Magnetic resonance imaging (MRI) provides a means of monitoring the change in position and the eventual breakdown of oil within sediments. The multidimensional technique allows the position of nuclei (most commonly protons) to be located within a known volume of substrate, e.g. sediment, hence offering a method of assessing the harming potential of oils in near-shore environments. Two-dimensional (2D) and three-dimensional (3D) MRI analyses of the measurement and movement of oil in estuarine sediments show that, using appropriate parameters, movement of the oil can be both observed and quantified. To aid the quantification a sample holder fabricated from polyvinylsiloxane, an inert material visible in magnetic resonance images has been used as an internal intensity standard. The results show the great potential of MRI in studying protonated contaminants in these materials, notwithstanding the presence of paramagnetic species in estuarine sediments which might distort the image. Sediments studied thus far have been collected from the Tay Estuary, Northeast Scotland.

Altritol synthesis by Notheia anomala.

The linear hexitol altritol has only been identified in six genera, all of them in the order Fucales of the brown algae. Five of these genera are closely related according to molecular phylogenetic and other data, while the sixth (Notheia) is an obligate epiphyte on two other altritol-containing genera with which it is symphanic. The possibility that Notheia obtains altritol from the algae on which it is epiphytic rather than by synthesizing altritol independently was investigated by supplying 13C-inorganic carbon in the light followed by mass spectrometric and nuclear magnetic resonance analysis. Notheia separated from the phorophyte Hormosira during exposure to 13C showed 13C enrichment in both altritol and mannitol, while the Hormosira only showed significant labelling of mannitol. These data show that altritol can be synthesized by Notheia, with implications for the number of gains and losses of the capacity to synthesize altritol in the evolution of the Fucales.

Nuclear magnetic resonance imaging (MRI) of diesel oil migration in estuarine sediment samples.

Magnetic resonance imaging (MRI) provides a means of monitoring the change in position and the eventual breakdown of oil within sediments. The multidimensional technique allows the position of nuclei (most commonly protons) to be located within a known volume of substrate, e.g. sediment, hence offering a method of assessing the harming potential of oils in near-shore environments. Two-dimensional (2D) and three-dimensional (3D) MRI analyses of the measurement and movement of oil in estuarine sediments show that, using appropriate parameters, movement of the oil can be both observed and quantified. To aid the quantification a sample holder fabricated from polyvinylsiloxane, an inert material visible in magnetic resonance images has been used as an internal intensity standard. The results show the great potential of MRI in studying protonated contaminants in these materials, notwithstanding the presence of paramagnetic species in estuarine sediments which might distort the image. Sediments studied thus far have been collected from the Tay Estuary, Northeast Scotland.

The relationship between microbial carbon and the resource quality of soil carbon.

The biological health of soil is an important aspect of soil quality because of the many critical functions performed by organisms in soil. Various indicators of soil quality have been proposed, but measurements of microbial biomass are most commonly used. During decomposition of plant residues in soil the relative intensities of the O-alkyl-C signal decreases and the alkyl-C signal increases in nuclear magnetic resonance (NMR) spectra. This leads to the suggestion that the alkyl-C to O-alkyl-C ratio of a soil may indicate the degree of decomposition. Consequently, the overall resource quality of soil C as a substrate for heterotrophic microorganisms may be inversely related to the alkyl-C to O-alkyl-C ratio. Our hypothesis is that a relationship exists between the size of the soil microbial community (microbial biomass) and the quality of soil carbon as a resource for microorganisms. New data have been combined with previously published data to show that there was a significant, negative correlation between the biomass C to total C (Cmic, to Corg) ratio and the alkyl-C to O-alkyl-C ratio (p < 0.01), which supports our hypothesis.

The application of magnetic resonance microimaging to the visible light curing of dental resins. Part 2. Dynamic imaging by the FLASH-MOVIE pulse sequence.

OBJECTIVES: To investigate the application of a rapid NMR imaging pulse sequence, FLASH-MOVIE, to the visible light curing of dental restorative materials. METHODS: The light guide was applied at one end of a cylindrical specimen of visible light curing unfilled resin and the light directed along the cylinder. During polymerisation an NMR imaging pulse sequence, FLASH-MOVIE, was run at 15s intervals with a 50 ms repetition time. The image of a 1mm thick vertical slice was recorded with a (125 microm)2 pixel size. RESULTS: Images with good contrast were obtained from all resin monomers. The image intensity from the polymer was indistinguishable from the background intensity. Thus, the progress of light activated polymerisation in the material could be followed in real time through a series of up to 16 images. Initially the image intensity increased in the material closest to the light guide, then decreased over time to zero. Concomitant with this fall, a "cure-front" moved through the specimen. SIGNIFICANCE: The FLASH-MOVIE NMR pulse sequence applied to microimaging of dental diacrylate resins can be used to obtain a dynamic record of visible light curing. A more refined experimental protocol will be required to apply this unique data to models proposed for this polymerisation mechanism.

Application of magnetic resonance microimaging to the study of dental caries.

Magnetic resonance microimaging (MRM) of teeth has continued to be developed. Two ways in which data can be presented have been investigated, 112-microm-thick image slices and pseudo-three-dimensional surface rendered images. Limitations of the latter have been demonstrated; the possible absence of structures having low intensity or incompleteness of the image at regions from which the signal intensity is low. This has implications for the MRM investigation of dental caries. However, all intensities are recorded and are available. Structures which give a low-intensity signal can be seen in image slices. MRM appears well suited to studying the development of dental caries, ideally in combination with other techniques. As MRM is non-destructive and non-invasive, it can be used in experiments which follow the progress of the disease, yet leaves the tissue intact for other investigations.

Solid state spatially resolved 1H and 19F nuclear magnetic resonance spectroscopy of dental materials by stray-field imaging.

As part of a program to evaluate the use of stray-field magnetic resonance microimaging (STRAFI) in dental materials research spatially resolved nuclear magnetic resonance (NMR) for solid dental cements has been investigated. By applying a quadrature echo pulse sequence to a specimen positioned in the stray-field of a NMR spectrometer superconducting magnet the magnetic resonance within a thin slice was obtained. The specimen was stepped through the field in 500 microm increments to record 1H and 19F profiles and T2 values at each point. The specimens were fully cured cylinders made from four types of restorative material (glass ionomer, resin modified glass ionomer, compomer, composite). The values for 1H T2 varied with material type and reflected the nature of the matrix structure. For all materials containing 19F in the glass two values were calculated for 19F T2, one short and one long. These were relatively invariant. Solid state magic angle spinning (MAS)-NMR showed that they came from the glass. This suggests that a proportion of the element is relatively mobile (in a glass phase) and the remainder is more tightly bound (in a compound dispersed in the glass). This demonstration, that NMR microimaging of both 1H and 19F in solid dental cements is possible, opens up exciting new possibilities for investigating the distribution of these elements (in particular fluorine) in solid dental materials.

Analysis of MRI images of a silicone elastomer under different axial loading conditions.

Silicone elastomers have been used as implants in orthopaedics for replacements of small joints such as the metacarpophalangeal joint. A series of experiments was conducted an axially tensioned silicone elastomer tendon spacer samples to determine whether there is a relationship between stress in the material and image intensity. With increasing load, reduced image intensities were observed using spin echo (SE), gradient echo (GE) and magnetic resonance imaging (MRI) snapshot techniques. MR attenuation was accentuated in the GE experiment. The T1 and T2 snapshot images were dim and showed a low signal-to-noise ratio, so analysis was limited. Changes in magnetic susceptibility are suggested as the mechanism causing reduced image intensity, due to the more pronounced attenuation in the GE image. An alternative mechanism is the alteration of crystallinity during loading, whereby molecular realignment modifies nuclear relaxation. The changes in spin lattice (T1) and spin spin (T2) relaxation times, which would support this, were not, however, satisfactorily demonstrated.

Application of 13C NMR to investigate the transformations and biodegradation of organic materials by wood- and soil-feeding termites, and a coprophagous litter-dwelling dipteran larva.

Solid-state 13C nuclear magnetic resonance spectroscopy has been used to characterize the C in samples of the food (wood), gut contents and faeces from the wood-feeding termite, Microcerotermes parvus; soil in the guts and mound material from the soil-feeding termite, Thoracotermes macrothorax; and the food and faeces from the litter-feeding, coprophagous larvae of the dipteran fly, Bibio marci. Spectra from the wood-feeding termite indicated preferential loss of polysaccharide and accumulation of lignin with some modification to the O-aromatic-C and methoxyl-C (O-methyl-C) components during passage through the gut. Spectra for the soil-feeding termite indicated little change in the distribution of 13C between resonances following passage through the gut, except for some evidence of preferential polysaccharide loss. Interpretation of the spectra from these organisms was restricted by the relatively low C content of the soils and mound material, and by the large contribution to the NMR spectra from the gut tissue rather than the gut contents. Spectra for the litter-feeding dipteran larvae indicated preferential feeding on the polysaccharide-rich component of the litter and then overall loss of polysaccharide-C and accumulation of both aromatic-C and methoxyl-C in the gut. These changes were greater for the second passage than for the first passage through the gut, suggesting that principally mechanical and physical changes occurred initially and that chemical digestion was prevalent during the second passage.

Magnetic resonance microimaging of carious teeth.

Three-dimensional (proton) magnetic resonance microimages of a number of extracted teeth (both healthy and carious) were obtained using a Bruker AM300WB magnetic resonance spectrometer fitted with a Bruker microimaging attachment. Full data sets have been acquired as 90-micron 3 voxels to enable high-quality full-surface (external and internal) reconstructions to be computed. Possession of such data has allowed mapping of the complete range of intensities in any selected image slice. The application of artificial color was found to aid the interpretation of both image types. All teeth showed the expected dental morphologies. The carious regions of the diseased teeth gave an intense image that was readily distinguishable from the other soft tissue.

An application of NMR microimaging to investigate nitrogen fixing root nodules.

Various techniques to obtain high-resolution NMR images (voxel size down to 39 x 39 x 250 microns) of nitrogen-fixing root nodules from soybean [Glycine max (Merr.)] and peanut (Arachis hypogaea L.) are compared. We describe the artefacts arising from changes in the magnetic susceptibility throughout the sample and how these can be minimised. A series of T1 (TR = 220 to 3020 ms) and T2 (TE = 9.3 to 33.6 ms) weighted images are presented. From these it has been possible to locate the oxygen diffusion barrier. A possible interpretation in terms of nodule biochemistry and physiology are given. The data and parameters presented are shown to serve as a basis for more extensive investigations of root nodules (e.g., the oxygen diffusion barrier or the mechanisms driving the regulation of the oxygen concentration in the infected zone by leghemoglobin) by NMR microimaging.

Non-destructive visualisation in three dimensions of freezing events in flowers of blackcurrant (Ribes nigrum L.) using spin-echo NMR microscopy.

Flowers of blackcurrant (Ribes nigrum L.) were subjected to freezing stress in vivo, and the resulting damage examined in three dimensions using a spin-echo nuclear magnetic resonance imaging sequence. Increased signal intensity was detected in the damaged flowers, particularly at the base of the style, in T2-weighted images. This is thought to be the result of intracellular freezing, which causes membrane damage and leakage of cellular contents. It is proposed that this represents the main site of damage within the flowers. The imaging of flowers of differing developmental ages showed larger increases in signal from fully open flowers after freezing damage compared with those in the initial stages of bud, suggesting that the enclosed nature of the flower buds may have a protective effect on the sensitive stylar base. The use of three-dimensional nuclear magnetic resonance imaging provides a rapid and effective means for the visualisation of freezing events within floral tissues; the effective resolution of the images enables greater accuracy and clarity in interpretation than hitherto possible in two dimensions.

High field NMR microscopic imaging of cultivated strawberry fruit.

The experimental conditions required for discrimination of various types of tissue in fruits of cultivated strawberry (Fragaria x Ananassa) at high fields (ca. 7 T) have been investigated. In marked contrast to soft fruits of other species, from which informative images have been derived at high fields using a variety of pulse sequences and acquisition parameters, appreciable image intensities from parenchymal and vascular tissues in healthy strawberry fruits were obtained only with a spin-echo imaging sequence using large sweep widths (ca. 100,000 Hz), and consequently small values for TE ( < 5 ms), indicating predominantly short T2 values for these tissues. Damage caused by infection by the fungal pathogen Botrytis cinerea is readily seen as a result of a large increase in T2 in the infected tissue, whereas ripening processes appear to be characterized primarily by small variations in the T2-weighted contrast and in the relative magnitudes of T1 between vascular and parenchymal tissue. In addition, it was possible selectively to enhance the contributions to images from the achenes ("seeds") by using very short relaxation delays, thereby enhancing T1-dominated contrast mechanisms.

Nuclear magnetic resonance microscopy of the development of the parasitoid wasp Venturia canescens within its host moth Plodia interpunctella.

Nuclear magnetic resonance microscopy was used to image the parasitoid wasp Venturia canescens (Hymenoptera: Ichneumonidae) within larval and pupal instars of its host, the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). The images were obtained using gradient-echo and chemical shift selective pulse sequences and clearly showed the location and shapes of the parasitoid as it developed from the L1 larva to a pupal stage within the host. The digestive, nervous, and tracheal systems of the host were identified and changes were observed as the host underwent metamorphosis. Destruction of the host tissues by the parasitoid was visible. It was found that the parasitoid first ate the fat body and digestive system of the host, allowing the host to continue to grow, and only progressed to the vital organs when its own development had neared pupation.

The chemical synthesis of Leishmania donovani phosphoglycan via polycondensation of a glycobiosyl hydrogenphosphonate monomer.

A polycondensation of 2,3,6-tri-O-benzoyl-4-O-(2,3,4-tri-O-benzoyl-beta-D-galactopyranosyl)-al pha-D-mannopyranosyl hydrogenphosphonate in the presence of trimethylacetyl chloride has been used to synthesize a linear poly[beta-D-galactopyranosyl-(1-->4)-alpha-D-mannopyranosyl phosphate] representing the phosphoglycan part of the lipophosphoglycan from Leishmania donovani.

Determination of the depth of cure for VLC composites by nuclear magnetic resonance microimaging.

OBJECTIVES: The purpose of this study was to evaluate the potential of nuclear magnetic resonance microimaging (NMRM) for investigating the depth of cure for visible light curing of dental composite materials. METHODS: Cylindrical composite specimens were light-cured within the NMRM instrument for predetermined times. Vertical slice, mid-resolution, spin-echo images were acquired using a Bruker AM300WB instrument with a Bruker microimaging attachment. Images were digitized and intensity profiles generated using deuterium oxide/water as a reference intensity. Separate specimens were made to obtain Vickers microhardness values as a function of depth to compare an established method for determining depth of cure with NMRM. RESULTS: A difference in NMR image intensity between uncured and cured composite resin has been detected. Values for integrated intensities were obtained at 150 microns intervals over the mid-fifth of the total image width. An abrupt transition was not seen at the cure front which advances with cumulative exposure time. NMRM produced similar data trends to microhardness measurement. SIGNIFICANCE: NMRM produces three-dimensional images of "mobile" hydrogen nuclei to a resolution of 10(-5) mm3. It is non-invasive, non-destructive and able to selectively image protons in different chemical environments. It can be used to investigate depth of cure for light curing materials, but must be regarded as a research technique and not one for routine measurement. Refinement is necessary, possible, and in progress to improve resolution from the present 10(-2) mm3 towards the limit, and to reduce noise.

The vascular architecture of the fruit receptacle of red raspberry determined by 3D NMR microscopy and surface-rendering techniques.

The three-dimensional structure of the vascular system in tht fruit receptacle of red raspberry was determined using NMR microscopy in combination with computer techniques which highlight the surfaces of specific tissue types. The surface-rendering technique is particularly valuable in situations, where there are large differences in image characteristics between the tissue of interest and the rest of the specimen, and thus ideal for the delineation of ilic vascular tissue in the raspberry receptacle. This was shown to consist of a conical network of bundles with a spiral pattern of gaps; tht carpellury traces emereed from the proximal end of each gap. The inner xylem and outer phloem tissues each appeared as a pair of fused columns in the surface-rendered images, and each carpellary trace had a separate supply from the xyiern and phloem.