Life Sciences Research for Lifelong Health

Publications david-oxley

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Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase.
EA Raiber, D Beraldi, G Ficz, HE Burgess, MR Branco, P Murat, D Oxley, MJ Booth, W Reik, S Balasubramanian

ABSTRACT: BACKGROUND: Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation. RESULTS: Our method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts. CONCLUSIONS: Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.

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Genome biology, 13, 8, R69, 2012

PMID: 22902005
DOI: 10.1186/gb-2012-13-8-r69

Open Access

The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r.
A Keniry, D Oxley, P Monnier, M Kyba, L Dandolo, G Smits, W Reik

The H19 large intergenic non-coding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extra-embryonic cell lineages, yet its physiological function is unknown. Here we show that miR-675, a microRNA (miRNA) embedded in H19's first exon, is expressed exclusively in the placenta from the gestational time point when placental growth normally ceases, and placentas that lack H19 continue to grow. Overexpression of miR-675 in a range of embryonic and extra-embryonic cell lines results in their reduced proliferation; targets of the miRNA are upregulated in the H19 null placenta, including the growth-promoting insulin-like growth factor 1 receptor (Igf1r) gene. Moreover, the excision of miR-675 from H19 is dynamically regulated by the stress-response RNA-binding protein HuR. These results suggest that H19's main physiological role is in limiting growth of the placenta before birth, by regulated processing of miR-675. The controlled release of miR-675 from H19 may also allow rapid inhibition of cell proliferation in response to cellular stress or oncogenic signals.

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Nature cell biology, 14, 7, 659-65, 2012

PMID: 22684254
DOI: 10.1038/ncb2521

Open Access

Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.
MJ Booth, MR Branco, G Ficz, D Oxley, F Krueger, W Reik, S Balasubramanian

5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.

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Science (New York, N.Y.), 336, 6083, 934-7, 2012

PMID: 22539555
DOI: 10.1126/science.1220671

Open Access

RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.
L Tavares, E Dimitrova, D Oxley, J Webster, R Poot, J Demmers, K Bezstarosti, S Taylor, H Ura, H Koide, A Wutz, M Vidal, S Elderkin, N Brockdorff

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

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Cell, 148, 4, 664-78, 2012

PMID: 22325148
DOI: 10.1016/j.cell.2011.12.029

Open Access

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.
MA Barber, A Hendrickx, M Beullens, H Ceulemans, D Oxley, S Thelen, M Thelen, M Bollen, HC Welch

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.

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The Biochemical journal, 443, 1, 173-83, 2012

PMID: 22242915
DOI: 10.1042/BJ20112078

Open Access

Protein identification by MALDI-TOF mass spectrometry.
Webster J, Oxley D

MALDI-TOF mass spectrometers are now commonplace and their relative ease of use means that most non-specialist labs can readily access the technology for the rapid and sensitive analysis of biomolecules. One of the main uses of MALDI-TOF-MS is in the identification of proteins, by peptide mass fingerprinting (PMF). Here we describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by 1D or 2D gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and destaining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF-MS of the tryptic peptides and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.

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Methods in molecular biology (Clifton, N.J.), 800, 1940-6029, 227-40, 2012

PMID: 21964792

CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL.
R Gilley, PA Lochhead, K Balmanno, D Oxley, J Clark, SJ Cook

The pro-apoptotic BH3 only protein BIM(EL) is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIM(EL) to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIM(EL), which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5-/- fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIM(EL) occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIM(EL) drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIM(EL) and may be relevant to the therapeutic use of agents such as paclitaxel.

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Cellular signalling, 24, 1, 170-80, 2012

PMID: 21924351
DOI: 10.1016/j.cellsig.2011.08.018

Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1.
SP Rowbotham, L Barki, A Neves-Costa, F Santos, W Dean, N Hawkes, P Choudhary, WR Will, J Webster, D Oxley, CM Green, P Varga-Weisz, JE Mermoud

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

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Molecular cell, 42, 3, 285-96, 2011

PMID: 21549307
DOI: 10.1016/j.molcel.2011.02.036

Open Access

PI3Kβ plays a critical role in neutrophil activation by immune complexes.
S Kulkarni, C Sitaru, Z Jakus, KE Anderson, G Damoulakis, K Davidson, M Hirose, J Juss, D Oxley, TA Chessa, F Ramadani, H Guillou, A Segonds-Pichon, A Fritsch, GE Jarvis, K Okkenhaug, R Ludwig, D Zillikens, A Mocsai, B Vanhaesebroeck, LR Stephens, PT Hawkins

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B₄. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

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Science signaling, 4, 168, ra23, 2011

PMID: 21487106
DOI: 10.1126/scisignal.2001617

Open Access

A novel foregut mucin characterized by a murine monoclonal autoantibody.
Binos S, Royce SG, Oxley D, Bacic A, Bhathal PS, Underwood JR

Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y antigens have been implicated in the induction of autoimmune gastritis. Monoclonal antibody D10 (D10 MAb) recognizes a highly conserved mucin expressed in the foregut of mammals and other vertebrates. The objective of this study was to biochemically characterize the autoantigen identified by D10 MAb and examine its autoimmunogenicity in the mouse. Characterization of the mucin autoantigen was undertaken following purification, by amino acid and carbohydrate analyses, deglycosylation, SDS-PAGE, and immunoblotting using D10 MAb. Autoimmune reactivity and specificity of D10 MAb were validated by immunohistochemistry and ELISA using mouse tissue. Induction of autoimmune gastritis was investigated following immunization of mice with D10 MAb-reactive heterologous mucin. D10 MAb was shown to be a murine anti-mucin autoantibody with a unique pattern of immunohistochemical staining of Brunner's glands of the duodenum and the cardiac glands, mucous neck cells, and pyloric glands of the stomach from inbred Balb/c mice in patterns identical to that previously reported in human tissue. Amino acid and carbohydrate analysis of purified D10 mucin reflected a compositional profile of a typical mucin molecule. Confirmation that D10 MAb recognizes a mucin was also provided by demonstration that the carbohydrate epitope resides on a high molecular weight (>1x10(6)Da), high-density (>1.40 g/mL) molecule comprised of greater than 60% carbohydrate. Mice immunized with D10 MAb-reactive, purified, heterologous mucin produced autoantibodies of identical specificity to the original D10 MAb. These data demonstrate the autoimmunogenic properties of a novel foregut mucin and raise the potential of anti-mucin autoantibodies in the induction of autoimmune gastritis.

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Hybridoma (2005), 29, 1557-8348, 87-100, 2010

PMID: 20455280

Fission yeast Iec1-ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism.
CJ Hogan, S Aligianni, M Durand-Dubief, J Persson, WR Will, J Webster, L Wheeler, CK Mathews, S Elderkin, D Oxley, K Ekwall, PD Varga-Weisz

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

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Molecular and cellular biology, 30, 3, 657-74, 2010

PMID: 19933844
DOI: 10.1128/MCB.01117-09

Open Access

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain.
D Corcos, MJ Osborn, LS Matheson, F Santos, X Zou, JA Smith, G Morgan, A Hutchings, M Hamon, D Oxley, M Brüggemann

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

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Blood, 115, 2, 282-8, 2010

PMID: 19822901
DOI: 10.1182/blood-2009-07-234864

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.
LS Matheson, MJ Osborn, JA Smith, D Corcos, M Hamon, R Chaouaf, J Coadwell, G Morgan, D Oxley, M Brüggemann

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

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International immunology, 21, 8, 957-66, 2009

PMID: 19561045
DOI: 10.1093/intimm/dxp062

Effect of enzymatic deimination on the conformation of recombinant prion protein.
DS Young, F Meersman, D Oxley, J Webster, AC Gill, I Bronstein, CR Lowe, DV Dear

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

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Biochimica et biophysica acta, 1794, 8, 1123-33, 2009

PMID: 19341825
DOI: 10.1016/j.bbapap.2009.03.013

The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins.
R Jones, PS James, D Oxley, J Coadwell, F Suzuki-Toyota, EA Howes

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

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Biology of reproduction, 79, 3, 421-31, 2008

PMID: 18448843
DOI: 10.1095/biolreprod.107.067314

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice.
X Zou, MJ Osborn, DJ Bolland, JA Smith, D Corcos, M Hamon, D Oxley, A Hutchings, G Morgan, F Santos, PJ Kilshaw, MJ Taussig, AE Corcoran, M Brüggemann

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.

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The Journal of experimental medicine, 204, 13, 3271-83, 2007

PMID: 18086860
DOI: 10.1084/jem.20071155

Open Access

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis.
Webster JM, Oxley D, Pettolino FA, Bacic A

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.

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Phytochemistry, 69, 0031-9422, 873-81, 2008

PMID: 18037144

Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues.
JT Huang, L Wang, S Prabakaran, M Wengenroth, HE Lockstone, D Koethe, CW Gerth, S Gross, D Schreiber, K Lilley, M Wayland, D Oxley, FM Leweke, S Bahn

Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (-35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (-30%, P=0.02) and RBCs (-60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (-18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (-35%, P=0.05; -51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.

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Molecular psychiatry, 13, 12, 1118-28, 2008

PMID: 17938634
DOI: 10.1038/sj.mp.4002108

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro.
DV Dear, DS Young, J Kazlauskaite, F Meersman, D Oxley, J Webster, TJ Pinheiro, AC Gill, I Bronstein, CR Lowe

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

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Biochimica et biophysica acta, 1774, 7, 792-802, 2007

PMID: 17572162
DOI: 10.1016/j.bbapap.2007.05.005

Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading.
S Kulkarni, KJ Woollard, S Thomas, D Oxley, SP Jackson

The ability of platelets to provide a highly reactive surface for the recruitment of other platelets and leukocytes to sites of vascular injury is critical for hemostasis, atherothrombosis, and a variety of inflammatory diseases. The mechanisms coordinating platelet-platelet and platelet-leukocyte interactions have been well defined and, in general, it is assumed that increased platelet activation correlates with enhanced reactivity toward other platelets and neutrophils. In the current study, we demonstrate a differential role for platelets in supporting platelet and neutrophil adhesive interactions under flow. We demonstrate that the conversion of spread platelets to microvesiculated procoagulant (annexin A5-positive [annexin A5+ve]) forms reduces platelet-platelet adhesion and leads to a paradoxical increase in neutrophil-platelet interaction. This enhancement in neutrophil adhesion and spreading is partially mediated by the proinflammatory lipid, platelet-activating factor (PAF). PAF production, unlike other neutrophil chemokines (IL-8, GRO-alpha, NAP-2, IL-1beta) is specifically and markedly up-regulated in annexin A5+ve cells. Physiologically, this spatially controlled production of PAF plays an important role in localizing neutrophils on the surface of thrombi. These studies define for the first time a specific proinflammatory function for annexin A5+ve platelets. Moreover, they demonstrate an important role for platelet-derived PAF in spatially regulating neutrophil adhesion under flow.

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Blood, 110, 6, 1879-86, 2007

PMID: 17548580
DOI: 10.1182/blood-2006-08-040980

Urinary pheromones promote ERK/Akt phosphorylation, regeneration and survival of vomeronasal (V2R) neurons.
Xia J, Sellers LA, Oxley D, Smith T, Emson P, Keverne EB

The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for Galpha(o) and V2R, whereas V1R and Galpha(i) immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked Galpha(o). Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation.

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The European journal of neuroscience, 24, 0953-816X, 3333-42, 2006

PMID: 17229082

DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH.
R Zhao, D Oxley, TS Smith, GA Follows, AR Green, DR Alexander

The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage-induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp(52)/iso-Asp(66) species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage-induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.

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PLoS biology, 5, 1, e1, 2007

PMID: 17177603
DOI: 10.1371/journal.pbio.0050001

Disease biomarkers in cerebrospinal fluid of patients with first-onset psychosis.
Huang JT, Leweke FM, Oxley D, Wang L, Harris N, Koethe D, Gerth CW, Nolden BM, Gr.oss S, Schreiber D, Reed B, Bahn S

Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods.

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PLoS medicine, 3, 1549-1676, e428, 2006

PMID: 17090210

Eukaryotic localization, activation and ubiquitinylation of a bacterial type III secreted toxin.
Stirling FR, Cuzick A, Kelly SM, Oxley D, Evans TJ

Type III secretion is a widespread method whereby Gram-negative bacteria introduce toxins into eukaryotic cells. These toxins mimic or subvert a normal cellular process by interacting with a specific target, although how toxins reach their site of action is unclear. We set out to investigate the intracellular localization of a type III toxin of Pseudomonas aeruginosa called ExoU, which has phospholipase activity and requires a eukaryotic factor for activity. We found that ExoU is localized to the plasma membrane and undergoes modification within the cell by addition of two ubiquitin molecules at lysine-178. A region of five amino acids at position 679-683 near the C-terminus of the ExoU protein controls both membrane localization and ubiquitinylation. Site-directed mutagenesis identified a tryptophan at position 681 as crucial for these effects. We found that the same region at position 679-683 was also required for cell toxicity produced by ExoU as well as in vitro phospholipase activity. Localization of the phospholipase ExoU to the plasma membrane is thus required for activation and allows efficient utilization of adjacent substrate phospholipids.

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Cellular microbiology, 8, 1462-5814, 1294-309, 2006

PMID: 16882033

Peptide mass fingerprinting: protein identification using MALDI-TOF mass spectrometry.
Webster J, Oxley D

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-staining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.

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Methods in molecular biology (Clifton, N.J.), 310, 1064-3745, 227-40, 2005

PMID: 16350956