Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome,... more Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insi...
The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed ... more The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMA...
Current human pluripotent stem cells lack the transcription factor circuitry that governs the gro... more Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propag...
Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated wi... more Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a singleN-glycosylation site (Asn-28) to which one of threeN-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that fou...
Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y ... more 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.
Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact... more Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact relationships have not been fully elucidated. The effect of varying blood flows on the accumulation of thrombotic material within arteries was investigated, with use of ...
The pro-survival protein Bcl-x L is critical for the resistance of tumour cells to DNA damage. We... more The pro-survival protein Bcl-x L 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 damageinduced Bcl-x L deamidation tightly correlates with T cell ...
Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass ... more Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosacchar...
Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namel... more Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: β-elimination (or desulfuration), which results on the loss of an H2S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH- anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electri...
1 Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom, 2 Department of... more 1 Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom, 2 Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany, 3 Proteomics Research Group, Babraham Institute, Cambridge, United Kingdom, 4 Ciphergen Biosystems Ltd., ...
Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysop... more Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.
Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome,... more Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insi...
The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed ... more The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMA...
Current human pluripotent stem cells lack the transcription factor circuitry that governs the gro... more Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propag...
Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated wi... more Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a singleN-glycosylation site (Asn-28) to which one of threeN-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that fou...
Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y ... more 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.
Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact... more Alterations in arterial blood flow are thought to predispose to thrombus formation, but the exact relationships have not been fully elucidated. The effect of varying blood flows on the accumulation of thrombotic material within arteries was investigated, with use of ...
The pro-survival protein Bcl-x L is critical for the resistance of tumour cells to DNA damage. We... more The pro-survival protein Bcl-x L 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 damageinduced Bcl-x L deamidation tightly correlates with T cell ...
Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass ... more Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosacchar...
Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namel... more Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: β-elimination (or desulfuration), which results on the loss of an H2S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH- anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electri...
1 Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom, 2 Department of... more 1 Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom, 2 Department of Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany, 3 Proteomics Research Group, Babraham Institute, Cambridge, United Kingdom, 4 Ciphergen Biosystems Ltd., ...
Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysop... more Autotaxin (ATX) the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA) is a secreted enzyme. Here we show that once secreted it can bind to the surface of cell secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interaction, ATX releases the LPA to activate cell surface G-protein coupled LPA receptors; inhibition of signaling by the receptor antagonist Ki1642 suggests these are either LPAR1 or LPAR3. The binding stimulates downstream signaling including AKT and MAPK phosphorylation, the release of intracellular stored calcium and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means whereby autotaxin-LPA signaling operates physiologically.
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Papers by David Oxley