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Biomedical subjects

P Taylor

Publications and source records attributed to P Taylor.

At least 271 records · Page 15Linked to original sources
PubMed1995

Theoretical analysis of the structure of the peptide fasciculin and its docking to acetylcholinesterase.

The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound fasciculin.

Acetylcholinesterase↗
PubMed1995

A case-control study of child bicycle injuries: relationship of risk to exposure.

In order to assess the relationship of the risk of injury requiring hospital attendance in children riding bicycles to sociodemographic factors and to measures of exposure, a population-based case-control study is being undertaken in a large area of suburban Melbourne, Australia. Particular attention is given to the measurement of individual exposure in several dimensions. Analysis of interim data from 109 cases and 118 controls shows that 51% of injuries occurred while the child was playing rather than making a trip on the bicycle and only 22% involved another vehicle. Boys used bicycles more commonly than girls but there was minimal evidence of an increased risk of injury in boys, adjusting for exposure. There was no evidence for an age trend in injury risk, but children from families in the lowest income category were at significantly increased risk. Exposure measures showed complex patterns of association with injury risk. Estimated time spent riding was more closely associated with risk than distance travelled, with an odds ratio of 2.2 (95% confidence interval 1.1-4.2) for children riding for more than 3 hours per week compared to children riding less than 1 hour. Riding more than 5 km on the sidewalk was also associated with increased risk (odds ratio 3.1, 95% CI 1.1-8.5). The elevated risk associated with sidewalk riding may be due to difficulties in negotiating uneven surfaces. The case-control study provides an ideal design for this type of investigation but valid and reliable measurement of exposure is difficult.

Accidents, Traffic↗
PubMed1995

Off to a rapid start: appraising maternal mortality and services.

Enthusiasm for 'doing something about Safe Motherhood' has been expressed in many developing countries, but priorities for action cannot be identified without adequately assessing a country's maternal mortality situation. It is also important, however, to avoid embarking on time-consuming research measuring indicators which are not essential to developing programs. After presenting an overview of ideal Safe Motherhood program components, the paper lists a series of questions which serve as an assessment tool for collecting useful information and for identifying data sources on maternal mortality and health. The framework for these questions centers around the following steps: (1) gaining an overview of health policy relevant to maternal mortality and morbidities; (2) assessing the magnitude and causes of maternal mortality and morbidity, and the characteristics of groups at particular risk; and (3) assessing the available inputs in terms of services (access, quality, providers, what is provided at various tiers, etc.) and in terms of the culture and existing resources and groups.

Developing Countries↗
PubMed1995

Structural bases for the specificity of cholinesterase catalysis and inhibition.

The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.

Animals↗
PubMed1995

Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity.

The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.

Amino Acid Sequence↗
PubMed1995

Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor.

Through specific intersubunit contacts, the four subunits of the nicotinic acetylcholine receptor assemble into an alpha 2 beta gamma delta pentamer. The specificity of subunit association leads to formation of proper ligand binding sites and to transport of assembled pentamers to the cell surface. To identify determinants of subunit association, we constructed chimeric subunits, transfected them into HEK 293 cells, and studied their association with wild-type subunits. We used beta gamma chimeras to determine sequences that associate with the alpha subunit to form a ligand binding site and found residues 21-131 of the gamma subunit sufficient to form the site. Residues 51-131 of the beta subunit do not form a binding site, but do promote surface expression of pentamers; of these residues, R117 is key for surface expression. We studied formation of tetramers by alpha and gamma subunits and dimers by alpha and delta subunits, and used gamma delta chimeras to identify sequences that result in either dimers or tetramers. The conserved residues I145 and T150 of the gamma subunit promote alpha gamma alpha gamma tetramer formation, whereas the corresponding residues in the delta subunit, K145 and K150, allow only alpha delta dimer formation.

Amino Acid Sequence↗
PubMed1995

Rehabits: a common language of functional assessment.

Probabilistic measurement models offered by Rasch and others can be used to link different functional assessment instruments into a single measurement system. This study assessed 54 subjects (diagnoses: 8 brain injuries, 7 neuromuscular, 22 musculoskeletal, 7 spinal cord, 10 stroke) admitted to a free-standing rehabilitation hospital at admission and discharge using both the Functional Independence Measure (FIM) and the Patient Evaluation and Conference System (PECS). Thirteen FIM and 22 PECS motor skills items were scaled together into a 35-item instrument, providing scale values for all items in the same unit of measurement. Separate FIM and PECS measures produced for each subject correlate .94 and .91 (p < .0001), respectively, with the cocalibration measures, and 0.91 (p < .0001) with each other. Either instrument's ratings are easily and quickly converted into the other's using the common unit of measurement, the rehabit (rehabilitation measuring unit). This article argues that the stability of the PECS and FIM item difficulty estimates over thousands of subjects, dozens of hospitals, hundreds of raters, and years of assessment is convincing evidence in support of the widespread use of their cocalibrated, common scale values as a functionometric ruler.

Calibration↗
PubMed1995

Determining the acuteness and stability of deep venous thrombosis by ultrasonic tissue characterization.

PURPOSE: The intent of the study was to determine whether ultrasonic tissue characterization (UTC) could indicate acuteness and stability of deep venous thrombosis (DVT) of the lower extremities. METHODS: Thrombi presenting as filling defects on color Doppler imaging in the common or superficial femoral or popliteal veins in 50 extremities in 45 patients with DVT were studied. Acute DVT was less than 4 days duration, and chronic DVT was greater than 21 days duration. UTC analysis of parameters from the normalized power spectrum of backscattered ultrasound signals from venous filling defects was performed. This spectrum approaches a straight line, and its basic parameters, slope, and Y-intercept are related to scatterer size, concentration, and the square of the scatterer-to-medium acoustic impedances. Ten of the DVT extremities were reexamined at 1 week to assess UTC changes that would indicate thrombus instability. RESULTS: Acute DVT (19 of the 50 extremities) could be distinguished from chronic DVT, mainly on the basis of significantly higher intercept values for the acute group, which were 11.6 relative decibels (dBr) higher than those of the chronic DVT group. Discriminant linear analysis of the two parameters indicated a sensitivity of 94.7% and specificity of 90.3% in correctly diagnosing acute DVT. In a small sample of 10 extremities reexamined at 1 week, acute DVT extremities showed a mean 9.4 dBr decrease in intercept values with no significant change in slope. CONCLUSIONS: UTC distinguished clinically defined acute from chronic DVT. In a small series of extremities, UTC revealed significant instability of acute thrombi in a selected patient population.

Acute Disease↗
PubMed1995

Patients with congenital myasthenia associated with end-plate acetylcholinesterase deficiency show normal sequence, mRNA splicing, and assembly of catalytic subunits.

A congenital myasthenic condition has been described in several patients characterized by a deficiency in end-plate acetylcholinesterase (AChE). The characteristic form of AChE in the end-plate basal lamina has the catalytic subunits disulfide linked to a collagen-like tail unit. Southern analysis of the gene encoding the catalytic subunits revealed no differences between patient and control DNA. Genomic DNA clones covering exon 4 and the alternatively spliced exons 5 and 6 were analyzed by nuclease protection and sequencing. Although allelic differences were detected between controls, we found no differences in exonic and intronic areas that might yield distinctive splicing patterns in patients and controls. The ACHE gene was cloned from genomic libraries from a patient and a control. Transfection of the cloned genes revealed identical species of mRNA and expressed AChE. Cotransfection of the genes expressing the catalytic subunits with a cDNA from Torpedo encoding the tail unit yielded asymmetric species that require assembly of catalytic subunits and tail unit. thus the catalytic subunits of AChE expressed in the congenital myasthenic syndrome appear identical in sequence, arise from similar splicing patterns, and assemble normally with a tail unit to form a heteromeric species.

Acetylcholinesterase↗
PubMed1995

Invited review: computer aids for decision-making in diagnostic radiology--a literature review.

This review looks at a variety of different ways in which computers can be used to assist in the interpretation of radiological images and in radiological decision-making. The issues involved in the design of computerized decision aids are introduced and four criteria proposed for evaluating such aids: need, practicality, veracity and relevance. These criteria are used to assess research into decision aids based on: image databases, numerical methods, expert systems, image processing and image understanding systems. Possible directions for research leading to aids of practical value are discussed in the conclusion.

Decision Making, Computer-Assisted↗
PubMed1994

Regulation of acetylcholinesterase mRNA stability by calcium during differentiation from myoblasts to myotubes.

The expression of acetylcholinesterase (AChE), nicotinic acetylcholine receptors (nAChR), and their corresponding mRNAs increases dramatically during the conversion of myoblasts to myotubes in C2-C12 cells. The increase in expression of nAChR arises from transcriptional activation of the genes encoding the receptor subunits, whereas stabilization of labile transcripts is primarily responsible for enhanced AChE expression. In a search for the signaling pathways responsible for stabilization of the AChE mRNA, we found that ryanodine, synthetic ryanodine receptor antagonists and L-type, but not N-type, Ca2+ channel blockers inhibit the differentiation-induced expression of AChE mRNA, but not the nAChR mRNA. Selective inhibition of increased expression of AChE is also evident. Inhibition by ryanodine and nifedipine is additive suggesting different target sites for the two Ca2+ channel ligands. Ryanodine binding sites can be detected in both myoblasts and myotubes, but they increase substantially during differentiation. Rates of AChE gene transcription are not altered by ryanodine and nifedipine, indicating that decreased Ca2+ availability prevents stabilization of the mRNA normally seen with differentiation. Muscle cells still undergo elongation and fusion in the presence of ryanodine or L-type Ca2+ channel antagonists. Ryanodine block is fully reversible, indicating functional integrity of the cellular expression system after the drug treatment. These findings indicate that intracellular ryanodine-sensitive calcium channels and extracellular L-type Ca2+ channels link to play an important role in stabilizing AChE mRNA and suggest that transient increases in intracellular Ca2+ may be critical for the commitment of AChE expression during myogenesis.

Acetylcholinesterase↗
PubMed1994

Site of fasciculin interaction with acetylcholinesterase.

Fasciculin, a 6750-Da peptide from the venom of Dendroaspis, is known to inhibit reversibly mammalian and fish acetylcholinesterases at picomolar concentrations, but is a relatively weak inhibitor of avian, reptile, and insect acetylcholinesterases and mammalian butyryl-cholinesterases. An examination of fasciculin association with several mutant forms of recombinant DNA-derived acetylcholinesterase from mouse shows that it interacts with a cluster of residues near the rim of the gorge on the enzyme. The aromatic residues, Trp286, Tyr72, and Tyr124, have the most marked influence on fasciculin binding, whereas Asp74, a charged residue in the vicinity of the binding site that affects the binding of low molecular weight inhibitors, has little influence on fasciculin binding. The 3 aromatic residues are unique to the susceptible acetylcholinesterases and, along with Asp74, constitute part of the peripheral anionic site. Fasciculin falls in the family of three-loop toxins that include the receptor blocking alpha-toxins and cardiotoxins. From this basic structural motif, a binding site has evolved on fasciculin to be highly specific for the peripheral site on acetylcholinesterase. Acetylthiocholine affects rates of fasciculin binding at concentrations causing substrate inhibition. In the case of the mutant cholinesterases where rates of fasciculin dissociation are more rapid, steady state kinetic parameters also show acetylthiocholine-fasciculin competition to be consistent with occupation at a peripheral or substrate inhibition site rather than the active center.

Acetylcholinesterase↗
PubMed1994

Conserved tyrosines in the alpha subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists.

Studies with site-directed labeling reagents have identified residues near the ligand binding pocket of the nicotinic acetylcholine receptor. Among these residues are three conserved tyrosines, Tyr-93, Tyr-190, and Tyr-198 of the alpha subunit. Previous studies combined mutagenesis, expression in Xenopus oocytes, and dose-response analysis to examine contributions of these tyrosines to agonist affinity. In this study, we prepared a series of mutants at each position, expressed them in 293 HEK cells, and studied binding of agonists and antagonists to mutant receptors on intact cells. We show that all three tyrosines contribute to binding of agonists, and that each tyrosine contributes roughly equally to the binding energy. Although the contributions are roughly equivalent, the nature of the contribution is not equivalent at each position. For Tyr-93 and Tyr-190 the aromatic hydroxyl is essential, whereas for Tyr-198 aromaticity of the side chain is essential. Nearly identical results were obtained for the elementary quaternary ligand tetramethylammonium, indicating that these tyrosines contribute to stabilization of the quaternary ammonium portion of agonist. Tyr-190 and Tyr-198 also contribute to binding of the competitive antagonist dimethyl-d-tubocurarine; the side chain specificity for binding supports tyrosine interactions with one of two quaternary ammonium groups in dimethyl-d-tubocurarine. Y190F, in addition to altering binding affinity, also affects the equilibrium between activatable and desensitized receptor states.

Animals↗
PubMed1994

Glycosylation sites selectively interfere with alpha-toxin binding to the nicotinic acetylcholine receptor.

Sequence analysis reveals unique features in the alpha-subunit of nicotinic acetylcholine receptors from the alpha-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-terminal to the Tyr190, Cys192-Cys193 region of the ligand binding domain, substitution of Trp187 and Phe189 by non-aromatic residues and alteration of the proline sequence Pro194-X-X-Pro197. Glycosylation signals were inserted into the toxin-sensitive mouse alpha-subunit by the mutations F189N and W187N/F189T. The F189N alpha-subunit, when transfected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in alpha-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11-fold reduction. The W187N mutant and the double mutant F189N/S191A lacking the requisite glycosylation signals exhibited little alteration in affinity, as did the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (alpha-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of alpha-conotoxin M1 have widely divergent dissociation constants of 2.1 and 14,800 nM. Expression of alpha/gamma- and alpha/delta-subunit pairs indicated that the high and low affinity sites are formed by the alpha/delta and alpha/gamma contacts, respectively.

Amino Acid Sequence↗
PubMed1994

Agonist-selective regulation of polyphosphoinositide metabolism in pulmonary artery smooth muscle cells.

Using a rat pulmonary artery smooth muscle cell line (PAC1), detailed analysis of polyphosphoinositide (PPI) metabolism reveals receptor type-selective patterns in the formation of inositol phosphates and 3-hydroxyphosphorylated PPIs. Responses to several agonists that stimulate hypertrophy or proliferation were examined, and distinct categories of response profile were observed. Thrombin and angiotensin II stimulated the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and the formation of several cytosolic species of inositol phosphates without the activation of PI 3-hydroxykinase. The response to thrombin was distinctive because a very large production of inositol 1,4-bisphosphate was accompanied by hydrolysis of PI 4-phosphate. The response to platelet-derived growth factor (PDGF) was distinguished by the production of the PI 3-hydroxykinase product, PI 3,4,5-trisphosphate, and the appearance of PI 3-hydroxykinase activity in immunoprecipitates. PDGF treatment of PAC1 cultures did not produce accumulation of detectable amounts of inositol 1,4,5-trisphosphate, although a small sustained elevation in the level of inositol monophosphate and a gradual accumulation of inositol 1,3,4-trisphosphate were observed. Characterization of these distinctive responses permitted us to correlate agonist-regulated PPI metabolism with induction of immediate-early genes and stimulation of hypertrophy or proliferation of PAC1 cultures (Rothman, A., Wolner, B., Button, D., and Taylor, P. (1994) J. Biol. Chem. 269, 6399-6404). Thrombin-stimulated PPI turnover and the production of a high level of inositol bisphosphate may be early signals linked to the induction of fosB and PAC1 cell hypertrophy, whereas the activation of PI 3-hydroxykinase and the accumulation of PI 3,4,5-trisphosphate in response to PDGF appear to be associated with mitogenesis.

Angiotensin II↗