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Biomedical subjects

P Taylor

Publications and source records attributed to P Taylor.

At least 127 records · Page 7Linked to original sources
PubMed1999

Biochemical and structural characterization of a divergent loop cyclophilin from Caenorhabditis elegans.

Cyclophilin 3 (CYP-3) is one of the most abundantly expressed cyclophilin isoforms in the free living nematode Caenorhabditis elegans. The detailed post-embryonic expression pattern of the cyp-3 transcript is unusual, peaking during early larval development. The spatial expression pattern was examined via reporter gene analysis demonstrating that the cyp-3 transcript is exclusively expressed in the single anterior excretory cell. Recombinant cyclophilin 3 has been purified, crystallized and solved to a resolution of 1.8 A. The peptidyl-prolyl isomerase activity of CYP-3 has been characterized against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 2.4 x 10(6) M(-1) s(-1). The immunosuppressive drug cyclosporin A binds and inhibits CYP-3 with an IC(50) value of 16 nM, comparable with the range of values found for human cyclophilin A. The x-ray structure shows that the overall fold and active site geometry is similar to other cyclophilin structures. There are however a number of distinctive features, and we use this structure and amino acid sequence alignment analysis to identify a subgroup of "divergent-loop cyclophilins". This subgroup has a number of uniquely conserved features: an additional loop between residues 48 and 54 (KSGKPLH); two cysteine residues (Cys(40) and Cys(168)) that are in close proximity but remain in the unoxidized form, and two other conserved residues, His(54) and Glu(83). We suggest that these features are functionally important for the role played by this class of cyclophilins during cellular responses to stress caused by changes in the redox environment or by up-regulation of cellular activity. This study represents a detailed biological, biochemical, and structural characterization of a single cyclophilin isoform in the model organism Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed1999

Results from the low level mercury sorbent test at the Oak Ridge Y-12 Plant in Tennessee.

A mercury sorbent test was performed near the headwaters of the Upper East Fork Poplar Creek at the Oak Ridge Y-12 Plant in Tennessee. The sorbents SIR-200 (ResinTech), Keyle:X (SolmeteX), and GT-73 (Rohm and Haas) were the best materials tested for low level mercury removal. Each of these sorbents has a thiol-based active site. None of the tested sorbents reduced the mercury concentration to less than the existing 12 ng/l NPDES limit. For this small scale test, SIR-200, Keyle:X, and GT-73 reduced the mercury concentration to less than 51 ng/l, which is a regulatory treatment goal. The other sorbents tested, including granular activated carbon (Filtrasorb 300), did not reduce the mercury concentration below 51 ng/l at any tested flow up to 5 bed volumes per minute. Because of the cost and large volume of sorbent, a wastewater treatment plant for this stream would be prohibitively expensive to construct and maintain.

Absorption↗
PubMed1999

Novel mechanism of inhibition of elastase by beta-lactams is defined by two inhibitor crystal complexes.

Two structurally related beta-lactams form different covalent complexes upon reaction with porcine elastase. The high resolution x-ray structures of these two complexes provide a clear insight into the mechanism of the reaction and suggest the design of a new class of serine protease inhibitors that resist enzyme reactivation by hydrolysis of the acyl intermediate. The presence of a hydroxyethyl substituent on the beta-lactam ring provides a new reaction pathway resulting in the elimination of the hydroxyethyl group and the formation of a stabilizing conjugated double bond system. In contrast, the presence of a diethyl substituent on the beta-lactam ring leads to addition of water. The two enzyme complexes show very different binding modes in the enzyme active site.

Animals↗
PubMed1999

Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin's disease.

DNA from malignant cells is present in the serum/plasma of cancer patients and DNA from this source is amenable to analysis by polymerase chain reaction (PCR). In the present study, we evaluated whether Epstein-Barr virus (EBV) DNA is present in the serum of patients with EBV-associated Hodgkin's disease (HD). Using conventional PCR, EBV DNA was detected in serum from 30/33 patients with EBV-associated HD but in only 6/26 patients with non-EBV-associated disease (p < 0.001). Samples from healthy individuals were negative and only 5/12 infectious mononucleosis samples were positive. Real-time quantitative PCR was subsequently employed to determine the concentration of EBV DNA present in serum; among positive samples the level ranged from 1 to 705 copies per 125 microliter of serum. Post-treatment samples from 5/14 cases with EBV-associated HD contained detectable EBV DNA; analysis of this small group of cases suggests that positivity in post-treatment samples correlates with risk factors indicative of a poor prognosis. Overall, our results are consistent with the notion that DNA from Reed-Sternberg cells is present in the serum of HD patients, and further suggest that serum EBV should be evaluated as a prognostic marker. Int. J. Cancer (Pred. Oncol.) 84:442-448, 1999.

Adult↗
PubMed1999

Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LüH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LüH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LüH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LüH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.

Acetylcholinesterase↗
PubMed1999

Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity.

We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.

Animals↗
PubMed1999

The influence of peripheral site ligands on the reaction of symmetric and chiral organophosphates with wildtype and mutant acetylcholinesterases.

The rates of inhibition of mouse acetylcholinesterase (AChE) (EC 3.1.1.7) by paraoxon, haloxon, DDVP, and enantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholines were measured in the presence and absence of AChE peripheral site inhibitors: gallamine, D-tubocurarine, propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolar concentrations enhanced the rates of inhibition by neutral organophosphorus compounds (OPs) while inhibition rates by cationic OPs were slowed down. When peripheral site ligand concentrations extended to millimolar, the extent of the enhancement decreased creating a bell shaped activation profile. Analysis of inhibition by DDVP and haloxon revealed that peripheral site inhibitors increased the second order reaction rates by increasing maximal rates of phosphylation.

Acetylation↗
PubMed1999

Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime.

We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LüH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LüH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LüH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LüH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LüH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.

Animals↗
PubMed1999

Knockout of one acetylcholinesterase allele in the mouse.

One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.

Acetylcholinesterase↗
PubMed1999

Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor.

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.

Amino Acid Substitution↗
PubMed1999

Crystal structure of mouse acetylcholinesterase. A peripheral site-occluding loop in a tetrameric assembly.

The crystal structure of mouse acetylcholinesterase at 2.9-A resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Omega loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking alpha-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops.

Acetylcholinesterase↗
PubMed1999

Detection of human retrovirus 5 in patients with arthritis and systemic lupus erythematosus.

OBJECTIVE: To examine whether human retrovirus 5 (HRV-5) infection is associated with autoimmune rheumatic disease. METHODS: DNA from patients with various disorders including inflammatory diseases and from normal subjects was tested by nested polymerase chain reaction (PCR) for HRV-5 proviral DNA. Positive results were confirmed by DNA sequencing. RESULTS: HRV-5 proviral DNA was detected in 53% of synovial samples from arthritic joints, in 12% of blood samples from patients with rheumatoid arthritis (RA), and in 16% of blood samples from patients with systemic lupus erythematosus. In contrast, it was not detectable by PCR of affected tissues from patients with several other autoimmune diseases and was found in only 1 of >200 tissue specimens obtained at autopsy from non-RA patients. Sequence analysis of the amplified viral segment showed genetic variation between samples with maintenance of the open reading frame, typical of a replicating infectious retrovirus. CONCLUSION: This is the first report of the frequent detection of HRV-5 in any disease. We propose that the possible involvement of HRV-5 in autoimmune and rheumatic disease should be investigated further.

Arthritis, Rheumatoid↗
PubMed1999

Crystallization and preliminary X-ray analysis of flavocytochrome c(3), the fumarate reductase from Shewanella frigidimarina.

The fumarate reductase (flavocytochrome c(3)) from Shewanella frigidimarina (formerly S. putrefaciens) NCIMB400 has been crystallized in the space group P2(1), with cell dimensions of a = 45.447 A, b = 92.107 A, c = 78.311 A, and beta = 91.038 degrees and one molecule per asymmetric unit. A native data set has been collected to 1.8 A. The gene encoding Fcc(3) from the S. frigidimarina type strain ACAM591 has been cloned and sequenced and the protein crystallized in space group P2(1) with cell dimensions of a = 45.359 A, b = 88.051 A, c = 77.473 A, and beta = 104.499 degrees. Anomalous data have also been collected from the NCIMB400 crystal allowing the heme iron positions to be identified.

Base Sequence↗
PubMed1999

Isolation and analysis of amplified cDNA fragments during detection of unknown polymorphisms with temperature modulated heteroduplex chromatography.

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated unit for high-throughput projects or manually when working with a few samples.

Chromatography↗
PubMed1999

Rigid videosigmoidoscopy vs conventional sigmoidoscopy. A randomized controlled study.

BACKGROUND: Not only is rigid sigmoidoscopy uncomfortable for patients, but visualisation of the rectosigmoid junction and sigmoid colon is successful in only 40-70% of examinations. A novel fine-bore rigid videosigmoidoscope is described and then compared with a rigid conventional sigmoidoscope for patient discomfort and length of insertion. METHOD: A total of 58 patients were examined with both sigmoidoscopes in a random order. Discomfort was scored on a visual analogue scale; length of insertion was scored by the surgeon. Patients were blinded to which sigmoidoscope was being used. The images from the video examination were transmitted in real time for a second opinion in a different hospital. RESULTS: The mean (SD) insertion distance of the videosigmoidoscope was 23.2 (5.9) cm, which was significantly further than with the conventional sigmoidosocpe 16.5 (3.8) cm (p < 0.01). The discomfort on a visual analogue score for the videosigmoidoscope was 3.0 (1.8), which was significantly less than for the conventional sigmoidoscope 5.5 (2.7) (p < 0.01). The five users of the equipment (four surgeons and one colorectal nurse practitioner) preferred the videosigmoidoscope for image quality and ease of examination. CONCLUSIONS: A thinner, longer, rigid videosigmoidoscope is a more effective means of looking at the proximal sigmoid colon. Despite being inserted further, it caused less discomfort than the conventional sigmoidoscope. High-quality video images can be recorded or transmitted for real-time teleconsultation.

Humans↗
PubMed1999

The development and evaluation of CADMIUM: a prototype system to assist in the interpretation of mammograms.

We have developed CADMIUM, a novel approach for the design of systems to assist in the interpretation of medical images. CADMIUM uses symbolic reasoning to relate information obtained from image processing to the decisions radiologists take. The approach is based on a symbolic decision procedure which has already been used successfully in a variety of nonimaging clinical decision systems. In CADMIUM this decision procedure is extended with models of three generic image interpretation tasks: detection, measurement and classification of image features. The extended procedure is used to construct the lines of reasoning needed in each task and to control the acquisition of information by image processing. CADMIUM has been evaluated as an aid to the differential diagnosis of microcalcifications on mammographic images. Radiographers who had been trained to interpret images performed better when using the advice provided by the system.

Artificial Intelligence↗