Biomedical subjects

P C Edwards

Publications and source records attributed to P C Edwards.

15 recordsLinked to original sources

PubMed2005

Sonic hedgehog gene-enhanced tissue engineering for bone regeneration.

Improved methods of bone regeneration are needed in the craniofacial rehabilitation of patients with significant bone deficits secondary to tumor resection, congenital deformities, and prior to prosthetic dental reconstruction. In this study, a gene-enhanced tissue-engineering approach was used to assess bone regenerative capacity of Sonic hedgehog (Shh)-transduced gingival fibroblasts, mesenchymal stem cells, and fat-derived cells delivered to rabbit cranial bone defects in an alginate/collagen matrix. Human Shh cDNA isolated from fetal lung tissue was cloned into the replication-incompetent retroviral expression vector LNCX, in which the murine leukemia virus retroviral LTR drives expression of the neomycin-resistance gene. The rat beta-actin enhancer/promoter complex was engineered to drive expression of Shh. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the transduced primary rabbit cell populations expressed Shh RNA. Shh protein secretion was confirmed by enzyme-linked immunosorbent assay (ELISA). Alginate/ type I collagen constructs containing 2 x 10(6) Shh-transduced cells were introduced into male New Zealand White rabbit calvarial defects (8 mm). A total of eight groups (N=6) were examined: unrestored empty defects, matrix alone, matrix plus the three cell populations transduced with both control and Shh expression vectors. The bone regenerative capacity of Shh gene enhanced cells was assessed grossly, radiographically and histologically at 6 and 12 weeks postimplantation. After 6 weeks, new full thickness bone was seen emanating directly from the alginate/collagen matrix in the Shh-transduced groups. Quantitative two-dimensional digital analysis of histological sections confirmed statistically significant (P<0.05) amounts of bone regeneration in all three Shh-enhanced groups compared to controls. Necropsy failed to demonstrate any evidence of treatment-related side effects. This is the first study to demonstrate that Shh delivery to bone defects, in this case through a novel gene-enhanced tissue-engineering approach, results in significant bone regeneration. This encourages further development of the Shh gene-enhanced tissue-engineering approach for bone regeneration.

Adipose Tissue↗

PubMed2000

ACX3, a novel medium-chain acyl-coenzyme A oxidase from Arabidopsis.

In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0- to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene.

Acyl-CoA Oxidase↗

PubMed1998

Characterisation of an improved two-dimensional p22121 crystal from bovine rhodopsin.

Dialysis of rhodopsin isolated from bovine rod outer segments resulted in the formation of a new two-dimensional crystal form suitable for electron crystallography. The crystals obtained were tubular or single layers and showed p22121 symmetry (a=60.6(+/-0.8) A, b=86.3(+/-1.6) A). For the first time the size and order of the crystals allowed us to take electron diffraction patterns showing spots to a resolution of about 3.5 A. Images were recorded at liquid nitrogen temperature using a high voltage field emission electron microscope. Out of a large number of images 20 crystalline areas were selected and processed with the MRC image processing software. A projection structure of bovine rhodopsin to 5 A resolution was calculated using amplitudes and phases extracted from these images. The achieved resolution exceeds the resolution of all previously obtained structures of frog, bovine and squid rhodopsin crystals. In this map small differences are observed compared to the previous maps. Helix 5 seems to be even more highly tilted and between the arc-shaped feature and helix 5 a peak is present suggesting that helix 3 is prolonging this feature towards helix 5. These observations are in agreement with the latest model for the three-dimensional arrangement of rhodopsin. The resolution achieved as well as the availability of electron diffraction data suggest that there is a good possibility to collect data from tilted crystals and calculate an improved three-dimensional structure of rhodopsin.

Animals↗

PubMed1996

Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines.

In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.

Alzheimer Disease↗

PubMed1996

Staging the pathological assembly of truncated tau protein into paired helical filaments in Alzheimer's disease.

Tau protein, which is incorporated into the core of paired helical filaments (PHFs) in Alzheimer's disease (AD), can be characterised immunochemically by C-terminal truncation at Glu-391 recognised by monoclonal antibody (mAb) 423, and acid-reversible occlusion of a generic tau epitope in the tandem repeat region recognised by mAb 7.51. PHFs are also characterised by the presence of binding sites for a fluorescent dye (thiazin red) which can be used to differentiate between amorphous and fibrillar states of tau and beta-amyloid proteins in AD. We have used double labelling confocal microscopy to investigate that state of aggregation of the tau antigens associated with the core structure of the PHF at early stages of neurofibrillary pathology. We report that the early abnormal tau deposits in cells vulnerable to neurofibrillary degeneration are characterised by C-terminal truncation at Glu-391, acid-reversible occlusion of the mAb 7.51 epitope, and the absence of binding sites for thiazin red, consistent with the amorphous non-fibrillar structure demonstrated by immunoelectron microscopy. Transition to the fibrillar state in the PHF is associated with acid-reversible occlusion of both mAb 7.51 and 423 epitopes, and acquisition of binding sites for thiazin red. In neurites, the transition between the two states of aggregation shows distal to proximal polarity, with the fibrillar state found nearest the cell body. These findings demonstrate that the assembly of tau protein into the PHF occurs in at least two stages, an amorphous stage characterised by C-terminal truncation and occlusion of sites within the tandem repeat region, and a fibrillar stage characterised by acid-reversible occlusion of both epitopes via addition of intact tau molecules in the fuzzy coat of the PHF.

Aged↗

PubMed1995

Alzheimer paired helical filaments, untreated and pronase digested, studied by vertical platinum-carbon replication and high resolution transmission electron microscopy.

Untreated paired helical filaments (PHF) and pronase treated PHF filaments have been stereoscopically imaged with a freeze-drying vertical platinum-carbon replication preparation method for TEM. The untreated PHF have an average wide region, W = 22.8 +/- 2.4 nm, a narrow region width, T = 10.6 +/- 1.7 nm, and a helical turn period, L = 78.6 +/- 13.4. The widths of the pronase treated PHF were significantly reduced and had average measurements of W = 14.8 +/- 1.2 nm, T = 5.7 +/- 1.0 nm, with the helical period unchanged, L = 75.4 +/- 17 nm. The surfaces of the untreated PHF contained approximately 1.0 and approximately 0.4 nm strands, the size of normal and denatured tau monomer. The pronase treated PHF contained approximately 1.0 and approximately 0.4 nm strands as well as approximately 2.0 nm strands. The stereoscopic images of the untreated and the pronase digested PHF do not support a double helical morphology for the PHF. The PHF appear to be long helical ribbons. The approximately 1.0 and approximately 2 nm substructure has been organized both parallel and orthogonal to the PHF-core axis for distances less than 80 nm. The most frequent structural appearance is of a disorganized PHF core. The surfaces of the untreated PHF also have a similar disorganized appearance.

Alzheimer Disease↗

PubMed1992

Molecular characterization and measurement of Alzheimer's disease pathology: implications for genetic and environmental aetiology.

The neuropathological changes seen in Alzheimer's disease represent an interaction between the ageing process in which normal intellectual function is retained, and changes which are specifically associated with severe cognitive deterioration. Molecular analysis of these changes has tended to emphasize the distinction between neurofibrillary pathology, which is intracellular and highly correlated with cognitive deterioration, and the changes associated with the deposition of extracellular amyloid, which appears to be widespread in normal ageing. Extracellular amyloid deposits consist of fibrils composed of a short 42 amino acid peptide (beta/A4) derived by abnormal proteolysis from a much larger precursor molecule (APP). The recent demonstration of a mutation associated with APP in rare cases with familial dementia, neurofibrillary pathology in the hippocampus and atypical cortical Lewy body pathology raises the possibility that abnormal processing of APP could be linked directly with neurofibrillary pathology. Neurofibrillary tangles and neuritic plaques are sites of dense accumulation of pathological paired helical filaments (PHFs) which are composed in part of an antigenically modified form of the microtubule-associated protein tau. The average brain tissue content of PHFs measured biochemically does not increase in the course of normal ageing but increases 10-fold relative to age-matched controls in patients with Alzheimer's disease. There is also a substantial (three-fold) disease-related decline in normal soluble tau protein relative to age-matched controls. This intracellular redistribution of a protein essential for microtubule stability in cortico-cortical association circuits may play an important part in the molecular pathogenesis of dementia in Alzheimer's disease. The role of abnormal proteolysis of APP in this process remains to be elucidated. Immunohistochemical studies on renal dialysis cases have failed to detect evidence of neurofibrillary pathology related to aluminium accumulation in brain tissue. Nevertheless it needs to be seen whether more sensitive biochemical assays of neurofibrillary pathology can demonstrate evidence of an association with aluminium.

Alzheimer Disease↗

PubMed1991

Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51.

The microtubule-associated protein tau that is incorporated into paired helical filaments (PHFs) undergoes some form of aberrant posttranslational processing in Alzheimer disease. Difficulties in deciding which changes are critical for PHF formation stem in part from the lack of immunochemical markers specific for PHF tau. The only monoclonal antibody (mAb) that is known to react with PHF tau but not with the predominant normal adult tau species is mAb 423. Another mAb (7.51, described in this paper) recognizes a segment of tau that is included in the minimal recognition unit required by mAb 423. Unlike 423, which is PHF tau-specific, mAb 7.51 recognizes all PHF core-derived tau as well as native soluble tau and recombinant tau expressed in bacteria and so serves as a generic tau marker. Both epitopes are in the 12-kDa fragment released from the Pronase-resistant core of the PHF (which encompasses the tandem repeat region). The mAb 7.51 epitope requires segments located in the last two repeats, which are common to all tau isoforms. The mAb 423 epitope requires sequences located near both the N and the C terminus of the 12-kDa fragment common to three- and four-repeat tau isoforms. Fragments denatured by concentrated formic acid and SDS regain 423 reactivity when denaturing agents are removed. Since the primary amino acid sequences of PHF tau and normal tau are identical in the repeat region, we conclude that 423 reactivity also requires a modification(s) occurring within an approximately 90-residue segment that are not present in tau proteins so far described in the human brain.

Alzheimer Disease↗

PubMed1991

Measurement of distinct immunochemical presentations of tau protein in Alzheimer disease.

The tau protein is a microtubule-associated protein that is normally located in nerve axons. In Alzheimer disease, it is a constituent of paired helical filaments (PHFs), which are the principal fibrous component of the characteristic neurofibrillary tangles. The tau protein, therefore, is abnormally sequestered in an insoluble form in PHFs in the cell body and dendrites in Alzheimer disease. We have used two monoclonal antibodies (mAbs) to selectively measure the levels of normal, soluble tau protein and of PHF-associated tau protein in the brain. mAb 423 binds to PHFs and recognizes a 12-kDa fragment of tau protein released by formic acid treatment of PHFs, but it does not recognize normal tau protein. In contrast, mAb 7.51 recognizes normal tau protein as well as the PHF core-derived tau fragment, but its epitope is concealed in the PHF-bound form. The differential binding properties for these two mAbs have enabled us in this study to quantify insoluble PHF-associated tau protein in the somatodendritic compartment as well as normal soluble tau protein in its predominantly axonal location. Our findings demonstrate that a distinct immunochemical presentation of tau protein recognized by mAb 423, a PHF-specific marker, can be used to quantify neurofibrillary pathology in Alzheimer disease independently of the presence of normal tau proteins.

Alzheimer Disease↗

PubMed1990

Competitive ELISA for the measurement of tau protein in Alzheimer's disease.

Tau protein is a major component of paired helical filaments (PHFs) which constitute the characteristic neurofibrillary tangle lesions observed in Alzheimer's disease. Two tau mAbs have been produced which show distinct patterns of immunoreactivity with intact human tau and with tau incorporated in PHFs. The mAb 423 recognises PHFs but not human tau on immunoblots whereas mAb 7/51 reacts with human tau but its epitope is buried within the PHF and is only exposed after formic acid treatment. A competitive ELISA has been developed for both of these mAbs and these have been used to quantify the two distinct tau epitopes in PHFs. Samples containing antigen are incubated with horseradish peroxidase-conjugated mAb at 4 degrees C for 16 h and non-adsorbed antibody then measured by binding, at 37 degrees C for 1 h, to a fragment of tau coated on microtitre plates. Bound enzyme-labelled antibody is measured kinetically using a spectrophotometer capable of automatically mixing the samples throughout a 2-min incubation with substrate and chromogen. The interfacing of the plate reader with a computer permits competitive curves to be plotted automatically using Softmax. Curves are fitted using a 4-parameter logistic algorithm which allows one to determine the relative immunoreactivity for different samples. The application of these assays to monitoring biochemical fractions and quantifying distinct immunochemical presentations of tau protein with these two mAbs is described.

Alzheimer Disease↗

PubMed1989

Nucleotide sequence of high-passage hepatitis A virus strain HM175: comparison with wild-type and cell culture-adapted strains.

The nucleotide sequence of cDNA from a high-passage, cell culture-adapted variant of hepatitis A virus strain HM175 was compared with the previously determined sequences of wild-type virus and two other cell culture-adapted variants. A total of 42 nucleotide changes were detected when the sequence was compared with wild-type virus. Five of these changes were common to all cell culture-adapted strains and a further two changes were shared by the strains that had experienced the greatest number of cell culture passages. The mutations were distributed throughout the genome coding for amino acid substitutions in regions 2B, 2C and 3D with silent changes in 1C and the 5' non-coding region. The possible relevance of these mutations to cell culture adaptation and attenuation is discussed.

Animals↗

PubMed1988

Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease.

A substantially enriched preparation of Alzheimer paired helical filaments (PHFs) has been used as a starting point for biochemical studies. Pronase treatment, which strips off adhering proteins, leaves a resistant core that is structurally intact. This has been used to raise a monoclonal antibody that decorates the filament core. The antibody has been used to follow the extraction of two peptide fragments (9.5 and 12 kDa) by immunoblotting. The link between the PHF as a morphological entity and these peptides has been established independently by photoaffinity labeling with a chemical ligand to the PHF core. Sequence analysis of these peptides was used to design oligonucleotide probes for cloning a cognate cDNA, which leads to its identification as human microtubule-associated tau protein. The sequencing of the 9.5- and 12-kDa peptides shows they are derived from a conserved region of tau containing three repeating segments. Since these fragments have been copurified with the Pronase-resistant core and are only released by subsequent steps, the corresponding part of the tau molecule must be tightly bound in the PHF core.

Alzheimer Disease↗

PubMed1988

Structural characterization of the core of the paired helical filament of Alzheimer disease.

The paired helical filament, the principal constituent of the neurofibrillary tangles characteristic of Alzheimer disease, is shown to consist of two structurally distinct parts. An external fuzzy region can be removed by Pronase treatment to leave a Pronase-resistant morphologically recognizable core. Scanning transmission electron microscopy gives an estimate for the mass per unit length as 79 kDa.nm-1 before Pronase treatment and 65 kDa.nm-1 after treatment. The fuzzy region carries all the epitopes recognized by two different antisera against microtubule-associated protein tau. By contrast, a monoclonal antibody (mAb) we have raised to paired helical filament cores (mAb 423) decorates Pronase-treated filaments much more strongly than it does untreated ones. We have shown in previous papers that the epitope recognized by mAb 423 is carried by a central 9.5-kDa fragment of tau protein, which therefore forms part of the Pronase-resistant core structure. The remainder of the tau protein incorporated into the filaments must contribute part, if not all, of the fuzzy region. The mass per unit length measurements imply that the three-domain structural subunit of the core that we visualized previously by image reconstruction has a molecular mass of approximately equal to 100 kDa.

Alzheimer Disease↗

PubMedDate not supplied

Examination of phosphorylated tau protein as a PHF-precursor at early stage Alzheimer's disease.

Hyperphosphorylated tau protein which can be isolated on the basis of insolubility in 1% sarkosyl (A68-tau fraction) is thought to represent a precursor pool for PHF assembly, associated histologically with neuritic pathology, which feeds into a more resistant tangle-associated PHF pool via cross-linking and proteolysis. We examined these predictions at the earliest detectable stages of neurofibrillary pathology. We report that there is no evidence that neuritic pathology represents an early pathologic stage, no evidence of an association between neuritic pathology and phosphorylated tau, no evidence of selective accumulation of phosphorylated tau at early stages of pathology, and no evidence for a precursor/product relationship between phosphorylated tau and PHFs during progression of pathology. We conclude that altered phosphorylation is a secondary process affecting 5% of PHFs and does not explain PHF assembly in Alzheimer's disease.

Aged↗

PubMedDate not supplied

Quantitative analysis of tau protein in paired helical filament preparations: implications for the role of tau protein phosphorylation in PHF assembly in Alzheimer's disease.

In Alzheimer's disease, there is a major redistribution of the tau protein pool from soluble to PHF-bound forms. PHF-bound tau can be distinguished from normal tau by acid reversible occlusion of a generic tau epitope in the tandem repeat region and characteristic sedimentation in the if-II protocol developed in this laboratory. We show that 85% of tau bound in the PHF-like configuration can be recovered in the if-II PHF-fraction. Less than 1% of this material was phosphorylated at the mAb AT8 site in aged clinical controls or in cases with minimal or mild dementia. Of tau phosphorylated at the mAb AT8 site, only 12% was found to co-sediment with PHFs. These low levels could not be explained by postmortem dephosphorylation. As more than 95% of PHF-tau is not phosphorylated, even at early stages of pathology, it is misleading to use the terms "PHF-tau" and "phosphorylated tau" as though they were synonymous, particularly as this implies a pathogenetic role which phosphorylation need not have.

Alzheimer Disease↗