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S J Davis

Publications and source records attributed to S J Davis.

At least 19 recordsLinked to original sources
PubMed2006

Eukaryotic expression: developments for structural proteomics.

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Animals↗
PubMed2006

Covering ENT out of hours: how confident are senior house officers?

BACKGROUND: The implementation of the European working time directive has led to an increase in cross-speciality out-of-hours cover. This survey illustrates ENT out-of-hours cover arrangements and assesses the implications for senior house officers (SHOs) responsible for managing emergencies. METHODS: A telephone survey of 100 ENT departments was conducted, asking the on-call SHO about departmental structure, on-call rota design, their previous ENT experience, access to SHO training and their confidence in managing emergencies. RESULTS: 44 per cent of departments used only ENT SHOs on the on-call rota. 73 per cent always had an ENT middle grade on call. In 60 per cent of hospitals, the ENT consultant was sometimes on call with only a non-ENT SHO. At the time of the study, 5 per cent of SHOs had no ENT experience, no access to training, were not confident in managing simple emergencies and were on-call without middle-grade cover. CONCLUSION: The current junior on-call structure for ENT has implications for patient management.

After-Hours Care↗
PubMed2005

PDCD1: a tissue-specific susceptibility locus for inherited inflammatory disorders.

Variation in genes encoding costimulatory molecules expressed on lymphocytes has been expected to contribute to the genetic component of inflammatory disease, but only the gene encoding the inhibitory protein, CTLA-4, seems consistently to confer disease susceptibility. Studies in murine models implicate the inhibitory product of the pd1 gene, programmed death-1, in the maintenance of peripheral tolerance to self-antigens. We identify 22 single-nucleotide polymorphisms (SNPs) in the equivalent human gene, PDCD1, a number of which show significant associations with the specific immunoglobulin E response to grass allergens in atopic individuals. Stepwise analyses indicate that four of the disease-associated SNPs have independent effects. The two most common haplotypes show positive and negative associations but rarer haplotypes are also likely to be of influence. In a case-control study, multiple regression analysis of genotypic data implies that PDCD1 also confers susceptibility to rheumatoid arthritis. Along with work linking PDCD1 with susceptibility to another autoimmune condition, systemic lupus erythematosus, our data identify PDCD1 as a second immunomodulatory gene with pleiotropic effects in human disease. Genes encoding negative regulators may generally confer a significant fraction of the genetic risk associated with inherited inflammatory disorders.

Alleles↗
PubMed2001

Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore.

Phytochromes comprise a principal family of red/far-red light sensors in plants. Although phytochromes were thought originally to be confined to photosynthetic organisms, we have recently detected phytochrome-like proteins in two heterotrophic eubacteria, Deinococcus radiodurans and Pseudomonas aeruginosa. Here we show that these form part of a widespread family of bacteriophytochromes (BphPs) with homology to two-component sensor histidine kinases. Whereas plant phytochromes use phytochromobilin as the chromophore, BphPs assemble with biliverdin, an immediate breakdown product of haem, to generate photochromic kinases that are modulated by red and far-red light. In some cases, a unique haem oxygenase responsible for the synthesis of biliverdin is part of the BphP operon. Co-expression of this oxygenase with a BphP apoprotein and a haem source is sufficient to assemble holo-BphP in vivo. Both their presence in many diverse bacteria and their simplified assembly with biliverdin suggest that BphPs are the progenitors of phytochrome-type photoreceptors.

Amino Acid Sequence↗
PubMed2001

Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses.

Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 A resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.

Abatacept↗
PubMed2001

Watching the hands of the Arabidopsis biological clock.

Oligonucleotide and cDNA microarrays have been used to analyse the mRNA levels of 8,000 genes in Arabidopsis thaliana throughout the day/night cycle. Genes involved in signal transduction and in various metabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light.

Arabidopsis↗
PubMed2001

CD2F-10: a new member of the CD2 subset of the immunoglobulin superfamily.

The CD2 subset of the immunoglobulin superfamily consists of a rapidly expanding family of leukocyte cell surface receptors, at least five of which (CD2, CD48, CD58, CD150, and CD244) are involved in lymphocyte activation as either receptors or ligands. Completion of the draft sequence of the human genome offers the possibility of systematically identifying the full set of proteins and interactions of this important family. Here we describe the identification and characterization of the first new member of the subset, CD2F-10, found exclusively by genome searching.

Amino Acid Sequence↗
PubMed2001

The immunological synapse and CD28-CD80 interactions.

According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR-MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR-MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28.

B7-1 Antigen↗
PubMed2001

The heme-oxygenase family required for phytochrome chromophore biosynthesis is necessary for proper photomorphogenesis in higher plants.

The committed step in the biosynthesis of the phytochrome chromophore phytochromobilin involves the oxidative cleavage of heme by a heme oxygenase (HO) to form biliverdin IXalpha. Through positional cloning of the photomorphogenic mutant hy1, the Arabidopsis HO (designated AtHO1) responsible for much of phytochromobilin synthesis recently was identified. Using the AtHO1 sequence, we identified families of HO genes in a number of plants that cluster into two subfamilies (HO1- and HO2-like). The tomato (Lycopersicon esculentum) yg-2 and Nicotiana plumbaginifolia pew1 photomorphogenic mutants are defective in specific HO genes. Phenotypic analysis of a T-DNA insertion mutant of Arabidopsis HO2 revealed that the second HO subfamily also contributes to phytochromobilin synthesis. Homozygous ho2-1 plants show decreased chlorophyll accumulation, reduced growth rate, accelerated flowering time, and reduced de-etiolation. A mixture of apo- and holo-phyA was detected in etiolated ho2-1 seedlings, suggesting that phytochromobilin is limiting in this mutant, even in the presence of functional AtHO1. The patterns of Arabidopsis HO1 and HO2 expression suggest that the products of both genes overlap temporally and spatially. Taken together, the family of HOs is important for phytochrome-mediated development in a number of plants and that each family member may uniquely contribute to the phytochromobilin pool needed to assemble holo-phytochromes.

Amino Acid Sequence↗
PubMed2001

Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1.

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Abatacept↗
PubMed2000

Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein.

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.

Animals↗
PubMed2000

Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity.

Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

Animals↗
PubMed2000

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2.

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Carrier Proteins↗
PubMed2000

LICOS, a primordial costimulatory ligand?

In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7-1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7-related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand.

Amino Acid Sequence↗
PubMed2000

Bacteriophytochromes: new tools for understanding phytochrome signal transduction.

The recent discovery of phytochrome-like photoreceptors, collectively called bacteriophytochromes, in a number of bacteria has greatly expanded our understanding of the origins and modes of action of phytochromes in higher plants. These primitive receptors contain an N-terminal domain homologous to the chromophore-binding pocket of phytochromes, and like phytochromes, they bind a variety of bilins to generate photochromic holoproteins. Following the chromophore pocket is a domain similar to two-component histidine kinases, suggesting that these bacterial photoreceptors function in phosphorelay cascades that respond to the light environment. Their organization and distribution support the views that higher-plant phytochromes evolved from a cyanobacterial precursor and that they act as light-regulated kinases. With the ability to exploit bacterial genetics, these bacteriophytochromes now offer simple models to help unravel the biochemical and biophysical events that initiate phytochrome signal transmission.

Amino Acid Sequence↗
PubMed2000

Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition.

T cell antigen recognition is accompanied by cytoskeletal polarization towards the APC and large-scale redistribution of cell surface molecules into 'supramolecular activation clusters' (SMACs), forming an organized contact interface termed the 'immunological synapse' (IS). Molecules are arranged in the IS in a micrometer scale bull's eye pattern with a central accumulation of TCR/peptide-MHC (the cSMAC) surrounded by a peripheral ring of adhesion molecules (the pSMAC). We propose that segregation of cell surface molecules on a much smaller scale initiates TCR triggering, which drives the formation of the IS by active transport processes. IS formation may function as a checkpoint for full T cell activation, integrating information on the presence and quality of TCR ligands and the nature and activation state of the APC.

Animals↗
PubMed2000

Structure and dimerization of a soluble form of B7-1.

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Amino Acid Sequence↗