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Biomedical subjects

Paul Taylor

Publications and source records attributed to Paul Taylor.

At least 37 records · Page 2Linked to original sources
PubMed2004

Enzymatic and structural characterization of non-peptide ligand-cyclophilin complexes.

Piperidine ligands are described that provide the first examples of non-peptidic ligand structures for the cyclophilin family of proteins. Crystal structures of two ligand complexes are compared with the unliganded protein and show ligand-induced changes in side-chain conformation and water binding. A peptidylprolyl cis-trans-isomerase assay showed the dissociation constants of the two ligands to be 320 and 25 mM. This study also provides the first published data for both enzymatic activity and three-dimensional structure for any protein-ligand complex that binds with a high-millimolar dissociation constant. The structures may be of relevance in the field of drug design, as they suggest starting points for the design of larger tighter-binding analogues.

Catalytic Domain↗
PubMed2004

Comparison of the ADVIA Centaur and Abbott AxSYM immunoassay systems for a routine diagnostic virology laboratory.

BACKGROUND: Routine diagnostic laboratories are confronted with an ever-increasing workload with limited resources. Automation has provided some solutions to these challenges particularly high through put analysers such as the Abbott AxSYM. OBJECTIVE: The aim of the present study was to compare the performance of two automated immunoassay systems in a diagnostic virology laboratory. Samples previously tested using the Abbott AxSYM were tested with a recently introduced immunoassay analyser, the Bayer ADVIA Centaur, for six virology analytes: HBsAg, anti-HBc total, anti-HBc IgM, anti-HBs, anti-HCV and anti-HIV1/O/2. STUDY DESIGN: This study was a retrospective analysis of stored serum samples previously tested on the Abbott AxSYM. Samples giving discrepant results were tested by other alternative immunoassays and re-tested on the Abbott AxSYM in the same freeze-thaw cycle. RESULTS: Although the sensitivities of the two automated immunoassays were similar there was improved specificity demonstrated for anti-HIV and anti-HCV when using the ADVIA Centaur assays. The low background signals allowed resolution of samples previously shown to have indeterminate results for anti-HIV antibody in the AxSYM assay. In addition, samples shown to have Abbott AxSYM anti-HCV results that could not be confirmed by RIBA and HCV molecular methods were shown to be negative by the ADVIA Centaur assay.

AIDS Serodiagnosis↗
PubMed2003

Visuomotor priming by pictures of hand postures: perspective matters.

Observing hand postures interacts with the preparation of similar actions. This may be due to motor encoding of the observed displays and/or to enhanced visual processing induced by motor planning. We studied the effects of the observer's perspective on motor representation, using a visuomotor priming task with simple responses. Participants were asked to grasp a bar in horizontal or vertical orientation. In Experiment 1, the prime stimuli were pictures of a hand in either "Own" or "Other perspective", and their orientation could be congruent or incongruent with the pre-specified grasping action. An overall effect of congruency was found, providing strong evidence for the automatic encoding of the primes. The effects of prime perspective were moderated by the availability of preview of the hand stimuli: with preview, congruency effects only occurred for "Own perspective" stimuli. Conversely, without preview, congruency effects were restricted to "Other perspective" primes. In Experiment 2, we replicated the "Own perspective advantage" with hand preview. In addition, we manipulated the stimulus onset asynchrony between prime stimulus and go-signal and found congruency effects to be restricted to the shorter asynchronies. We interpret the "Own perspective advantage" as the result of an enhancement of action relevance of the prime stimuli during the preview interval, driven by motor planning. In contrast, we explain the "Other perspective advantage" as a stimulus-driven visuo-motor effect, based on more frequent experience with suddenly appearing hands of conspecifics than with suddenly appearing own body parts.

Adult↗
PubMed2003

Collapsing glomerulopathy coexisting with membranous glomerulonephritis in native kidney biopsies: a report of 3 HIV-negative patients.

Collapsing glomerulopathy (CG), a variant of idiopathic focal segmental glomerulosclerosis (FSGS), can occur in both human immunodeficiency virus (HIV)-positive and HIV-negative patients. Idiopathic membranous glomerulonephritis (MGN) has been reported to coexist with FSGS, but rarely with CG. We report 3 HIV-negative patients (2 men, 1 woman) who developed nephrotic syndrome secondary to MGN complicated by CG, with relatively rapid disease progression despite aggressive therapy.

Adult↗
PubMed2003

Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop.

A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis. Two distinct binding modes are observed, one of which resembles the hydrogen bonding pattern of bound ATP. In the second binding mode, the ligands trigger a conformational change in the activation T loop by inducing movement of Lys(33) and Asp(145) side chains. The family of molecules discovered provides an excellent starting point for the design and synthesis of tight binding inhibitors, which may lead to a new class of antiproliferative drugs.

Adenosine Triphosphate↗
PubMed2003

Insights into antifolate resistance from malarial DHFR-TS structures.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.

Amino Acid Sequence↗
PubMed2003

Proteomics in Vaccinology and Immunobiology: An Informatics Perspective of the Immunone.

The postgenomic era, as manifest, inter alia, by proteomics, offers unparalleled opportunities for the efficient discovery of safe, efficacious, and novel subunit vaccines targeting a tranche of modern major diseases. A negative corollary of this opportunity is the risk of becoming overwhelmed by this embarrassment of riches. Informatics techniques, working to address issues of both data management and through prediction to shortcut the experimental process, can be of enormous benefit in leveraging the proteomic revolution. In this disquisition, we evaluate proteomic approaches to the discovery of subunit vaccines, focussing on viral, bacterial, fungal, and parasite systems. We also adumbrate the impact that proteomic analysis of host-pathogen interactions can have. Finally, we review relevant methods to the prediction of immunome, with special emphasis on quantitative methods, and the subcellular localization of proteins within bacteria.

Journal Article↗
PubMed2003

Assessment of benefit in tele-ophthalmology using a consensus panel.

A videoconferencing link was established from a hospital in South Africa to Moorfields Eye Hospital in London. A clinician in South Africa used a video slit-lamp and videoconferencing equipment to capture images and communicate with a specialist in London. Over 12 months, 113 cases were discussed in teleconsultations. Case-notes were subsequently obtained for 90 cases (80%). Three consultant ophthalmologists took part in the consensus process, one from the UK and two from South Africa. The consensus panel achieved on average 78-96% agreement on the items rated. In approximately half the cases, the panel judged that teleconsultation had had an effect on diagnosis. In nine cases (10%) there was potential for definite improvement in visual health as a result of tele-ophthalmology and in a further 48 cases (53%) there was potential for possible improvement in visual health. The consensus process identified 35 cases (39%) where there was potential improvement in general health as a result of tele-ophthalmology. Despite the methodological limitations of our approach, it was possible to learn a great deal about the effectiveness of the telemedicine intervention.

Adolescent↗
PubMed2003

Structures of immunophilins and their ligand complexes.

This review includes an analysis of available X-ray and NMR structures of both members of the immunophilin family; cyclophilins and the FK-506 binding proteins (FKBPs). Available structures are compared and contrasted to highlight different structural features seen both within and between species. Each immunophilin family has been structurally characterised with a variety of small molecule ligands, principally immunosuppressive drugs and their analogues and an overview of these complexes is also presented. Currently the Protein Data Base contains over 60 entries for cyclophilins and over 40 entries for FKBPs. A number of FKBP related structures are also available including structures of MIP (Macrophage Infectivity Potentiator protein) from Legionella pneumophila and Trypanosoma cruzi and Trigger Factor from Mycoplasma genitalium. For all structures discussed in the review a summary of the available biological data is also presented.

Amino Acid Sequence↗
PubMed2003

Intravenous ascorbic acid: protocol for its application and use.

High dose intravenous(i.v.) ascorbic acid (AA) has been used as therapy for infectious disease from bacterial and viral origin and adjuvant therapy for cancer. In this publication we describe a clinical protocol that has been developed over the past twenty years utilizing high dose i.v. AA as therapy for cancer. This includes principles of treatment, rationale, baseline workup, infusion protocol, precautions and side effects.

Anti-Infective Agents↗
PubMed2002

Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1.

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.

Amino Acid Sequence↗
PubMed2002

Structural and biological characterisation of the gut-associated cyclophilin B isoforms from Caenorhabditis elegans.

The free-living nematode Caenorhabditis elegans expresses 18 cyclophilin isoforms, eight of which are conserved single domain forms, comprising two closely related secreted or type B forms (CYP-5 and CYP-6). Recombinant CYP-5 has been purified, crystallised and the X-ray structure solved to a resolution of 1.75A. The detailed molecular architecture most strongly resembles the structure of human cyclophilin B with conserved changes in loop structure and N and C-terminal extensions. Interestingly, the active site pocket is occupied by a molecule of dithiothreitol though this has little effect on the geometry of the active site which is similar to other cyclophilin structures. The peptidyl-prolyl isomerase activity of CYP-5 has been characterised against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 3.6x10(6)M(-1)s(-1) that compares with a value of 6.3x10(6)M(-1)s(-1) for human cyclophilin B. The immunosuppressive drug cyclosporin A binds and inhibits CYP-5 with an IC(50) value of 50nM, which is comparable to the value of 84nM found for human cyclophilin B. CYP-6 has 67% sequence identity with CYP-5 and a molecular model was built based on the CYP-5 crystal structure. The model shows that CYP-5 and CYP-6 are likely to have very similar structures, but with a markedly increased number of negative charges distributed around the surface of CYP-6. The spatial expression patterns of the cyclophilin B isoforms were examined using transgenic animals carrying a LacZ reporter fusion to these genes, and both cyp-5 and cyp-6 are found to be expressed in an overlapping fashion in the nematode gut. The temporal expression pattern of cyp-5 was further determined and revealed a constitutive expression pattern, with highest abundance levels being found in the embryo.

Amino Acid Sequence↗
PubMed2002

Homologues of human macrophage migration inhibitory factor from a parasitic nematode. Gene cloning, protein activity, and crystal structure.

Cytokines are the molecular messengers of the vertebrate immune system, coordinating the local and systemic immune responses to infective organisms. We report here functional and structural data on cytokine-like proteins from a eukaryotic pathogen. Two homologues of the human cytokine macrophage migration inhibitory factor (MIF) have been isolated from the parasitic nematode Brugia malayi. Both molecules (Bm-MIF-1 and Bm-MIF-2) show parallel functions to human MIF. They are chemotactic for human monocytes and activate them to produce IL-8, TNF-alpha, and endogenous MIF. The human and nematode MIF homologues share a tautomerase enzyme activity, which is in each case abolished by the mutation of the N-terminal proline residue. The crystal structure of Bm-MIF-2 at 1.8-A resolution has been determined, revealing a trimeric assembly with an inner pore created by beta-stranded sheets from each subunit. Both biological activity and crystal structure reveal remarkable conservation between a human cytokine and its parasite counterpart despite the considerable phylogenetic divide among these organisms. The strength of the similarity implies that MIF-mediated pathways play an important role in nematode immune evasion strategies.

Amino Acid Sequence↗
PubMed2002

A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90.

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.

Amino Acid Motifs↗
PubMed2002

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.

The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

Amino Acid Sequence↗
PubMed2002

An implantable two channel drop foot stimulator: initial clinical results.

This article reports preliminary results of pilot studies of a new implantable two channel drop foot stimulator. The stimulator consists of an externally worn transmitter inductively coupled to an implanted receiver unit located in the lower leg, lateral and distal to the knee. The receiver is connected to electrodes located under the epineurium of the deep and the superficial peroneal nerves. Stimulation is triggered by detection of heel lift and terminated at heel strike in a manner similar to surface mounted systems. The location of the electrodes allows for a degree of selectivity over the resultant moment about the ankle joint that is not possible with surface stimulation of the common peroneal nerve. The two subjects used the stimulator on a regular basis and showed increases in walking speed of between 10% and 44% when compared to their baseline measurements. Isometric tests have demonstrated that the stimulator allows selective and repeatable stimulation of ankle joint muscles.

Electric Stimulation Therapy↗
PubMed2001

Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable lipase from Bacillus stearothermophilus P1.

The gene encoding a thermostable lipase secreted by Bacillus stearothermophilus P1 has been cloned and overexpressed in Escherichia coli. The recombinant lipase was purified to homogeneity using ammonium sulfate precipitation, anion-exchange chromatography (Poros 20 HQ) and Sephacryl S-200HR. The molecular mass was shown to be 43 209 Da by mass spectrometry. Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitating agent. Determination of the structure by molecular replacement with existing mesophilic lipase structures has proved unrewarding, as there is less than 20% sequence identity with known lipase structures, but preliminary results with heavy-atom soaking indicate that this strategy will allow the structure to be solved. The availability of this new lipase structure will be of particular significance because it will be the first thermostable lipase to be described.

Amino Acid Sequence↗