Biomedical subjects

D J Read

Publications and source records attributed to D J Read.

At least 19 recordsLinked to original sources

PubMed2005

Small angle neutron scattering observation of chain retraction after a large step deformation.

The process of retraction in entangled linear chains after a fast nonlinear stretch was detected from time-resolved but quenched small angle neutron scattering (SANS) experiments on long, well-entangled polyisoprene chains. The statically obtained SANS data cover the relevant time regime for retraction, and they provide a direct, microscopic verification of this nonlinear process as predicted by the tube model. Clear, quantitative agreement is found with recent theories of contour length fluctuations and convective constraint release, using parameters obtained mainly from linear rheology. The theory captures the full range of scattering vectors once the crossover to fluctuations on length scales below the tube diameter is accounted for.

Journal Article↗

PubMed2003

Neutron-mapping polymer flow: scattering, flow visualization, and molecular theory.

Flows of complex fluids need to be understood at both macroscopic and molecular scales, because it is the macroscopic response that controls the fluid behavior, but the molecular scale that ultimately gives rise to rheological and solid-state properties. Here the flow field of an entangled polymer melt through an extended contraction, typical of many polymer processes, is imaged optically and by small-angle neutron scattering. The dual-probe technique samples both the macroscopic stress field in the flow and the microscopic configuration of the polymer molecules at selected points. The results are compared with a recent "tube model" molecular theory of entangled melt flow that is able to calculate both the stress and the single-chain structure factor from first principles. The combined action of the three fundamental entangled processes of reptation, contour length fluctuation, and convective constraint release is essential to account quantitatively for the rich rheological behavior. The multiscale approach unearths a new feature: Orientation at the length scale of the entire chain decays considerably more slowly than at the smaller entanglement length.

Journal Article↗

PubMed2002

Theoretical and finite-element investigation of the mechanical response of spinodal structures.

In recent years there have been major advances in our understanding of the mechanisms of phase separation in polymer and copolymer blends, to the extent that good control of phase-separated morphology is a real possibility. Many groups are studying the computational simulation of polymer phase separation. In the light of this, we are exploring methods which will give insight into the mechanical response of multiphase polymers. We present preliminary results from a process which allows the production of a two-dimensional finite-element mesh from the contouring of simulated composition data. We examine the stretching of two-phase structures obtained from a simulation of linear Cahn-Hilliard spinodal phase separation. In the simulations, we assume one phase to be hard, and the other soft, such that the shear modulus ratio G is large (>or= 10(3)). We indicate the effect of varying composition on the material modulus and on the distribution of strains through the stretched material. We also examine in some detail the symmetric structures obtained at 50% composition, in which both phases are at a percolation threshold. Inspired by simulation results for the deformation of these structures, we construct a "scaling" theory, which reproduces the main features of the deformation. Of particular interest is the emergence of a lengthscale, below which the deformation is non-affine. This length is proportional to G(1/4), and hence is still quite small for all reasonable values of this ratio. The same theory predicts that the effective composite modulus scales also as G(1/4), , which is supported by the simulations.

Journal Article↗

PubMed2001

Exploitation of pollen by mycorrhizal mycelial systems with special reference to nutrient recycling in boreal forests.

Very large quantities of pollen are released annually by wind-pollinated trees, which dominate northern forest ecosystems. Since pollen is enriched in both nitrogen and phosphorus, this recurrent pulse of deposition constitutes a significant potential source of these elements in what are known to be severely nutrient-limited systems. Here, we demonstrate for the first time, to our knowledge, that an ectomycorrhizal fungus, Paxillus involutus, is able to scavenge effectively for nitrogen and phosphorus in pollen and to return a significant proportion of each nutrient to its autotrophic host, Betula pendula. More than 75 and 96%, respectively, of the nitrogen and phosphorus were removed from pollen in microcosms containing the mycorrhizal fungus, 29 and 25%, respectively, being transferred to the plants. In contrast, in microcosms without the mycorrhizal fungus only 42 and 35%, respectively, of nitrogen and phosphorus were lost from the pollen, presumably as a result of export by saprotrophs, and only 12 and 7%, respectively, were transferred to the plants. We hypothesize that this process of resource recapture, by contributing significantly to the ability of the trees to sustain the necessary annual investment in pollen production, will have a major impact upon their reproductive capabilities and hence 'fitness'.

Ecosystem↗

PubMed2001

Large-scale forest girdling shows that current photosynthesis drives soil respiration.

The respiratory activities of plant roots, of their mycorrhizal fungi and of the free-living microbial heterotrophs (decomposers) in soils are significant components of the global carbon balance, but their relative contributions remain uncertain. To separate mycorrhizal root respiration from heterotrophic respiration in aboreal pine forest, we conducted a large-scale tree-girdling experiment, comprising 9 plots each containing about 120 trees. Tree-girdling involves stripping the stem bark to the depth of the current xylem at breast height terminating the supply of current photosynthates to roots and their mycorrhizal fungi without physically disturbing the delicate root-microbe-soil system. Here we report that girdling reduced soil respiration within 1-2 months by about 54% relative to respiration on ungirdled control plots, and that decreases of up to 37% were detected within 5 days. These values clearly show that the flux of current assimilates to roots is a key driver of soil respiration; they are conservative estimates of root respiration, however, because girdling increased the use of starch reserves in the roots. Our results indicate that models of soil respiration should incorporate measures of photosynthesis and of seasonal patterns of photosynthate allocation to roots.

Carbon↗

PubMed2001

Rates and quantities of carbon flux to ectomycorrhizal mycelium following 14C pulse labeling of Pinus sylvestris seedlings: effects of litter patches and interaction with a wood-decomposer fungus.

We used a novel digital autoradiographic technique that enabled, for the first time, simultaneous visualization and quantification of spatial and temporal changes in carbon allocation patterns in ectomycorrhizal mycelia. Mycorrhizal plants of Pinus sylvestris L. were grown in microcosms containing non-sterile peat. The time course and spatial distribution of carbon allocation by P. sylvestris to mycelia of its mycorrhizal partners, Paxillus involutus (Batsch) Fr. and Suillus bovinus (L.): Kuntze, were quantified following 14C pulse labeling of the plants. Litter patches were used to investigate the effects of nutrient resource quality on carbon allocation. The wood-decomposer fungus Phanerochaete velutina (D.C.: Pers.) Parmasto was introduced to evaluate competitive and territorial interactions between its mycelial cords and the mycelial system of S. bovinus. Growth of ectomycorrhizal mycelium was stimulated in the litter patches. Nearly 60% of the C transferred from host plant to external mycorrhizal mycelium (> 2 mm from root surfaces) was allocated to mycelium in the patches, which comprised only 12% of the soil area available for mycelial colonization. Mycelia in the litter patch most recently colonized by mycorrhizal mycelium received the largest investment of carbon, amounting to 27 to 50% of the total 14C in external mycorrhizal mycelium. The amount of C transfer to external mycelium of S. bovinus following pulse labeling was reduced from a maximum of 167 nmol in systems with no saprotroph to a maximum of 61 nmol in systems interacting with P. velutina. The 14C content of S. bovinus mycelium reached a maximum 24-36 h after labeling in control microcosms, but allocation did not reach a peak until 56 h after labeling, when S. bovinus interacted with mycelium of P. velutina. The mycelium of S. bovinus contained 9% of the total 14C in the plants (including mycorrhizae) at the end of the experiment, but this was reduced to 4% in the presence of P. velutina. The results demonstrate the dynamic manner in which mycorrhizal mycelia deploy C when foraging for nutrients. The inhibitory effect of the wood-decomposer fungus P. velutina on C allocation to external mycorrhizal mycelium has important implications for nutrient cycling in forest ecosystems.

Autoradiography↗

PubMed2000

Symbiotic fungal associations in 'lower' land plants.

An analysis of the current state of knowledge of symbiotic fungal associations in 'lower' plants is provided. Three fungal phyla, the Zygomycota, Ascomycota and Basidiomycota, are involved in forming these associations, each producing a distinctive suite of structural features in well-defined groups of 'lower' plants. Among the 'lower' plants only mosses and Equisetum appear to lack one or other of these types of association. The salient features of the symbioses produced by each fungal group are described and the relationships between these associations and those formed by the same or related fungi in 'higher' plants are discussed. Particular consideration is given to the question of the extent to which root fungus associations in 'lower' plants are analogous to 'mycorrhizas' of 'higher' plants and the need for analysis of the functional attributes of these symbioses is stressed. Zygomycetous fungi colonize a wide range of extant lower land plants (hornworts, many hepatics, lycopods, Ophioglossales, Psilotales and Gleicheniaceae), where they often produce structures analogous to those seen in the vesicular-arbuscular (VA) mycorrhizas of higher plants, which are formed by members of the order Glomales. A preponderance of associations of this kind is in accordance with palaeohbotanical and molecular evidence indicating that glomalean fungi produced the archetypal symbioses with the first plants to emerge on to land. It is shown, probably for the first time, that glomalean fungi forming typical VA mycorrhiza with a higher plant (Plantago lanceolata) can colonize a thalloid liverwort (Pellia epiphylla), producing arbuscules and vesicles in the hepatic. The extent to which these associations, which are structurally analogous to mycorrhizas, have similar functions remains to be evaluated. Ascomycetous associations are found in a relatively small number of families of leafy liverworts. The structural features of the fungal colonization of rhizoids and underground axes of these plants are similar to those seen in mycorrhizal associations of ericaceous plants like Vaccinium. Cross inoculation experiments have confirmed that a typical mycorrhizal endophyte of ericaceous plants, Hymenoscyphus ericae, will form associations in liverworts which are structurally identical to those seen in nature. Again, the functional significance of these associations remains to be examined. Some members of the Jungermanniales and Metzgeriales form associations with basidiomycetous fungi. These produce intracellular coils of hyphae, which are similar to the pelotons seen in orchid mycorrhizas, which also involve basidiomycetes. The fungal associates of the autotrophic Aneura and of its heterotrophic relative Cryptothallus mirabilis have been isolated. In the latter case it has been shown that the fungal symbiont is an ectomycorrhizal associate of Betula, suggesting that the apparently obligate nature of the association between the hepatic and Betula in nature is based upon requirement for this particular heterotroph.

Fungi↗

PubMed1999

Plant diversity and productivity experiments in european grasslands

At eight European field sites, the impact of loss of plant diversity on primary productivity was simulated by synthesizing grassland communities with different numbers of plant species. Results differed in detail at each location, but there was an overall log-linear reduction of average aboveground biomass with loss of species. For a given number of species, communities with fewer functional groups were less productive. These diversity effects occurred along with differences associated with species composition and geographic location. Niche complementarity and positive species interactions appear to play a role in generating diversity-productivity relationships within sites in addition to sampling from the species pool.

Journal Article↗

PubMed1999

Molecular cloning of neuropathy target esterase (NTE).

Covalent modification of NTE, a neuronal protein with serine esterase activity, by certain organophosphates (OP) initiates degeneration of long axons in the peripheral and central nervous system. Simple inhibition of NTE esterase activity does not initiate neuropathy; the latter requires aging of the OP bound to the catalytic serine residue so that a negatively-charged species is left attached to the active site. This may indicate that a non-esterase function of NTE is important for axonal maintenance. We have recently cloned NTE and shown that it is unrelated to any known serine hydrolases but contains a novel C-terminal domain which is conserved from bacteria to man. Furthermore, the catalytic serine is located within this domain at the centre of a helical hydrophobic segment of the polypeptide's secondary structure. The integrity of NTE would be severely compromised by the presence of a negatively-charged organophosphate moiety at this site. Implications for possible higher-order structures and functions for NTE are discussed.

Binding Sites↗

PubMed1998

Neuropathy target esterase and a homologous Drosophila neurodegeneration-associated mutant protein contain a novel domain conserved from bacteria to man.

The N-terminal amino acid sequences of proteolytic fragments of neuropathy target esterase (NTE), covalently labelled on its active-site serine by a biotinylated organophosphorus ester, were determined and used to deduce the location of this serine residue and to initiate cloning of its cDNA. A putative NTE clone, isolated from a human foetal brain cDNA library, encoded a 1327 residue polypeptide with no homology to any known serine esterases or proteases. The active-site serine of NTE (Ser-966) lay in the centre of a predicted hydrophobic helix within a 200-amino-acid C-terminal domain with marked similarity to conceptual proteins in bacteria, yeast and nematodes; these proteins may comprise a novel family of potential serine hydrolases. The Swiss Cheese protein which, when mutated, leads to widespread cell death in Drosophila brain [Kretzschmar, Hasan, Sharma, Heisenberg and Benzer (1997) J. Neurosci. 17, 7425-7432], was strikingly homologous to NTE, suggesting that genetically altered NTE may be involved in human neurodegenerative disease.

Amino Acid Sequence↗

PubMed1998

Neuropathy target esterase: immunolocalization to neuronal cell bodies and axons.

Determination of the molecular mechanisms involved in organophosphate-induced axonopathy may help to elucidate those involved in normal axonal maintenance and in other neurodegenerative conditions. In this study we aimed to define the cellular distribution of neuropathy target esterase, the primary target protein for neuropathic organophosphates. A synthetic peptide corresponding to the sequence of a proteolytic fragment of neuropathy target esterase purified from chicken brain was used to raise a rabbit antiserum designated R28. The antiserum was shown by immunoprecipitation and western blotting of brain extracts to react with a polypeptide of the expected molecular size (155,000 mol. wt); this reaction was blocked by preincubating the antiserum with the immunizing peptide. Prominent intracellular immunostaining by R28 was seen in neuronal cell bodies and, in some cases, proximal axon segments in frozen sections of chicken brain cortex, optic tectum, cerebellum, spinal cord, and dorsal root ganglia. Cells with glial morphology were not immunostained, neither were normal sciatic nerve or motor end plates. However, 8-12 h following sciatic nerve ligation, immunoreactive material was seen to accumulate both proximal and, to a lesser extent, distal to the ligature, indicating that neuropathy target esterase undergoes fast axonal transport. No gross qualitative or quantitative changes in the above pattern of neuropathy target esterase immunoreactivity were detected in tissue obtained from chickens one or three days following treatment with a neuropathic organophosphate. The presence of neuropathy target esterase in essentially all neurons indicates that the selective vulnerability of long axons to neuropathic organophosphates is dependent on factors additional to the presence of the target protein.

Amino Acid Sequence↗

PubMed1994

Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase.

We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.

Animals↗

PubMed1993

Stereo-specific degradation of the R-(+) isomer of O-n-hexyl-S-methylphosphorothioamidate catalysed by rabbit serum.

Resolved isomers of O-n-hexyl-S-methylphosphorothioamidate (HXM) which had been synthesised by separate stereospecific routes were analysed by chiral glc: about 2-3% of R-(+) isomer was found in the S-(-) sample and accounted for nearly all the inhibitory power against neuropathy target esterase. Incubation of racemic HXM with rabbit serum led to slow but very specific disposal of R-(+) isomer to undetectable levels with very slight loss of S-(-): the rate of disposal was roughly estimated to be about 1% of the published rate of hydrolysis of paraoxon. Incubation with crystalline chymotrypsin caused a preferential but not totally selective disposal of S-(-) isomer.

Animals↗

PubMed1993

Screening of O-ethyl O-4-nitrophenyl phosphoramidate (ENPP) for delayed neuropathic potential.

O-Ethyl-O-4-nitrophenylphosphoramidate is a short-acting anticholinesterase and a possible candidate for a prophylactic agent against nerve agents since human acetylcholinesterase inhibited by this agent undergoes rapid spontaneous reactivation which can be accelerated further, if necessary, by treatment with oximes. Doses of the agent > 1 mg/kg (s.c.) given to unprotected rats were fatal in a short time but 2 rats and one hen given 0.5 mg/kg survived. Hens given 2.5 or 4 mg/kg s.c. 20 min after prophylactic physostigmine + atropine survived acute effects and were killed 4.5 or 24 h later. Brain and spinal cord neuropathy target esterase levels of these hens were depressed only 4-10% compared with levels in brains from hens given only oxime + atropine or of undosed animals. Clinical signs of neuropathy were not seen in surviving birds observed for 3 weeks. It appears there would be negligible delayed neuropathic hazard associated with administration of O-ethyl-O-4-nitrophenylphosphoramidate at subacute doses.

Animals↗

PubMed1993

Prophylaxis against and promotion of organophosphate-induced delayed neuropathy by phenyl di-n-pentylphosphinate.

Phenyl di-n-pentylphosphinate (PPP) is a potent inhibitor of neuropathy target esterase (NTE) with negligible effect on acetylcholinesterase: I50S at 37 degrees C for 20 min and pH 8, respectively are 0.2 microM and > 2mM. PPP is not neuropathic. This is compatible with the fact that inhibited NTE is autopsy material from hens dosed with PPP can always be reactivated in vitro, presumably because no 'aging' reaction has occurred. PPP (10 mg/kg s.c.) given to hens up to 4 days before severely neuropathic doses (1.7 mg/kg) of diisopropylphosphorofluoridate (DFP) prevented neuropathic but not cholinergic effects of DFP. Hens given PPP 3 days after a sub-neuropathic dose of DFP (0.4 mg/kg) developed severe clinical neuropathy (clinical scores of 7 and 5 compared with DFP-plus-solvent scores 0,1,3). These prophylactic and promoting effects are similar to those exerted by phenylmethanesulphonyl fluoride (PMSF) at doses which inhibit NTE. In 3 out of 4 birds a pre-dose with PMSF (15 mg/kg) prevented the promoting effect of 120 mg/kg PMSF given after DFP.

Animals↗

PubMed1992

Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.

Animals↗

PubMed1991

Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homologues.

The interaction with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) in vivo of methamidophos (O,S-dimethyl phosphorothioamidate), its resolved stereoisomers and five higher O-alkyl homologues has been examined along with the ability of these compounds to cause organophosphorus-induced delayed polyneuropathy (OPIDP) in adult hens. For the lower homologues AChE was more sensitive than NTE and it was impossible to achieve high inhibition of NTE in vivo without both prophylaxis and therapy against acute anticholinesterase effects; for the n-hexyl homologue high inhibition of NTE could be achieved without obvious anticholinesterase effects and spontaneous reactivation of inhibited AChE was seen as in vitro. The maximum tolerated dose of L(-) methamidophos or of the ethyl or iso-propyl homologues did not inhibit NTE more than 60%, and surviving birds did not develop OPIDP. The n-propyl, n-butyl and n-hexyl compounds caused typical OPIDP at doses causing a peak of 70-95% inhibition of NTE in brain, spinal cord and sciatic nerve soon after dosing. Racemic methamidophos caused unusually mild OPIDP associated with very high inhibition of NTE at doses estimated to be greater than 8 times the unprotected LD50 and the D-(+) isomer caused OPIDP at about 5-7 x LD50. Clinical effects correlated with histopathology in 19 out of 20 examined birds. In contrast to results of many previous studies with organophosphates and phosphonates, all these cases of OPIDP were associated with formation of inhibited NTE which could be reactivated ex vivo by treatment of autopsy tissue with KF solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1989

Biochemical and clinical tests of the delayed neuropathic potential of some O-alkyl O-dichlorophenyl phosphoramidate analogues of methamidophos (O,S-dimethyl phosphorothioamidate).

The interaction in vivo of four O-alkyl O-2,5-dichlorophenyl phosphoramidates with neural neuropathy target esterase (NTE) and acetylcholinesterase (AChE) and their ability to cause delayed polyneuropathy in hens has been examined. Previous studies in vitro (Vilanova, Johnson & Vicedo, Pestic. Biochem. Physiol., 28 (1987) 224) had led to the prediction that these compounds would not be neuropathic but, rather, would be prophylactic agents against organophosphorus-induced delayed polyneuropathy. In vivo the effects of these esters on the enzymes differ in 2 respects from effects in vitro: (i) Relative sensitivity of the enzymes was different: thus greater than 50% of brain NTE remained 24 h after an oral dose of 15 mg/kg of the n-hexyl ester while only 10-30% of AChE remained although NTE was the more sensitive enzyme in vitro; (ii) In no case could the inhibited NTE or AChE in autopsy samples from birds dosed with any of the 4 esters be reactivated by treatment with potassium fluoride in vitro: the inhibited enzymes produced by incubation of tissue with the esters in vitro had been reactivatable. Prophylaxis, with therapy in some cases, was required to prevent acute anticholinesterase poisoning when doses were sufficient to cause high inhibition of neural NTE. Inhibition in brain was typically 5-10% more than in spinal cord and 10-15% more than in sciatic nerve. Unambiguous signs of polyneuropathy (Grade 3 or more on an 8-point scale) were not seen in birds observed up to 3 weeks after doses which caused less than 70% inhibition of NTE in brain and spinal cord or less than 60% inhibition in sciatic nerve of pair-dosed birds assayed 24 h after dosing. Doses of 300, 10, 100 and 65 mg/kg, respectively, of the methyl, ethyl, n-butyl and n-hexyl esters caused greater than 70% inhibition of NTE in all 3 neural tissues and neuropathy in the majority of observed birds. Analysis of consolidated dose/response data from 36 assayed and 51 observed birds showed that effects of Grade 3 or more were produced in about 90% of birds when inhibition of NTE was greater than 90% in brain, greater than 85% in spinal cord or greater than 75% in sciatic nerve.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗