Life Sciences Research for Lifelong Health

Publications

The Babraham Institute Publications database contains details of all publications resulting from our research groups and scientific services.

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Title / Authors / Details Open Access Download

The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice.
W Mi, B Beirowski, TH Gillingwater, R Adalbert, D Wagner, D Grumme, H Osaka, L Conforti, S Arnhold, K Addicks, K Wada, RR Ribchester, MP Coleman

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

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Brain : a journal of neurology, 128, Pt 2, 405-16, 2005

PMID: 15644421

DOI: 10.1093/brain/awh368


Open Access

The two-domain hypothesis in Beckwith-Wiedemann syndrome: autonomous imprinting of the telomeric domain of the distal chromosome 7 cluster.
Cerrato F, Sparago A, Di Matteo I, Zou X, Dean W, Sasaki H, Smith P, Genesio R, Bruggemann M, Reik W, Riccio A

A large cluster of imprinted genes is located on the mouse distal chromosome 7. This cluster is well conserved in humans and its dysregulation results in the overgrowth- and tumour-associated Beckwith-Wiedemann syndrome. Two imprinting centres (IC1 and IC2) controlling different sets of genes have been identified in the cluster, raising the hypothesis that the cluster is divided into two functionally independent domains. However, the mechanisms by which imprinting of genes in the IC2 domain (e.g. Cdkn1c and Kcnq1) is regulated have not been well defined, and recent evidence indicates that distantly located cis-acting elements are required for IC2 imprinting. We show that the maternal germ-line methylation at IC2 and the imprinted expression of five genes of the IC2 domain are correctly reproduced on an 800 kb YAC transgene when transferred outside of their normal chromosomal context. These results, together with previous transgenic studies, locate key imprinting control elements within a 400 kb region centromeric of IC2 and demonstrate that each of the two domains of the cluster contains the cis-acting elements required for the imprinting control of its own genes. Finally, maternal, but not paternal, transmission of the transgene results in fetal growth restriction, suggesting that during evolution the acquisition of imprinting may have been facilitated by the opposite effects of the two domains on embryo growth.

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Human molecular genetics, 14, 0964-6906, 503-11, 2005

PMID: 15640248


Open Access

The neutrophil: the unnoticed threat in xenotransplantation?
LA Cardozo, DB Rouw, LR Ambrose, M Midulla, O Florey, DO Haskard, AN Warrens

Xenotransplantation offers one way to circumvent the widening gap between the demand for and supply of human organs for transplantation, and the pig is widely regarded as the donor animal most likely to prove appropriate. Most attention has focused on the adaptive immune response to xenogeneic tissue. However, there is optimism that it may soon be possible to overcome that hurdle. In this paper, we consider the possibility of the direct recognition of xenogeneic tissue by neutrophils.

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Transplantation, 78, 12, 1721-8, 2004

PMID: 15614144


A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction.
P Perry, S Sauer, N Billon, WD Richardson, M Spivakov, G Warnes, FJ Livesey, M Merkenschlager, AG Fisher, V Azuara

Mammalian embryonic stem (ES) cells can either self-renew or generate progenitor cells that have a more restricted developmental potential. This provides an important model system to ask how pluripotency, cell commitment and differentiation are regulated at the level of chromatin-based changes that distinguish stem cells from their differentiated progeny. Here we show that the differentiation of ES cells to neural progenitors results in dynamic changes in the epigenetic status of multiple genes that encode transcription factors critical for early embryonic development or lineage specification. In particular, we demonstrate that DNA replication at a subset of neural-associated genes including Pax3, Pax6, Irx3, Nkx2.9 and Mash1 is advanced upon neural induction, consistent with increased locus accessibility. Conversely, many ES-associated genes including Oct4, Nanog, Utf1, Foxd3, Cripto and Rex1 that replicate early in ES cells switch their replication timing to later in S-phase in response to differentiation. Detailed analysis of the Rex1 locus reveals that delayed replication extends to a 2.8 Mb region surrounding the gene and is associated with substantial reductions in the level of histone H3K9 and H4 acetylation at the promoter. These results show that loss of pluripotency (and lineage choice) is associated with extensive and predictable changes in the replication timing of key regulator genes.

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Cell cycle (Georgetown, Tex.), 3, 12, 1645-50, 2004

PMID: 15611653


Open Access

CD28 regulates the translation of Bcl-xL via the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway.
Wu LX, La Rose J, Chen L, Neale C, Mak T, Okkenhaug K, Wange R, Rottapel R

In concert with the TCR, CD28 promotes T cell survival by regulating the expression of the antiapoptotic protein Bcl-x(L). The mechanism by which CD28 mediates the induction of Bcl-x(L) remains unknown. We show that although signaling through the TCR is sufficient to stimulate transcription of Bcl-x(L) mRNA, CD28, by activating PI3K and mammalian target of rapamycin, provides a critical signal that regulates the translation of Bcl-x(L) transcripts. We observe that CD28 induced 4E-binding protein-1 phosphorylation, an inhibitor of the translational machinery, and that CD28 costimulation directly augmented the translation of a Bcl-x(L) 5'-untranslated region reporter construct. Lastly, costimulation by CD28 shifted the distribution of Bcl-x(L) mRNA transcripts from the pretranslation complex to the translationally active polyribosomes. These results demonstrate that CD28 relieves the translational inhibition of Bcl-x(L) in a PI3K/mammalian target of rapamycin-dependent manner.

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Journal of immunology (Baltimore, Md. : 1950), 174, 0022-1767, 180-94, 2005

PMID: 15611240


Open Access

Simulated diffusion of phosphorylated CheY through the cytoplasm of Escherichia coli.
K Lipkow, SS Andrews, D Bray

We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis. A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With Smoldyn, we constructed a three-dimensional model of an E. coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.

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Journal of bacteriology, 187, 1, 45-53, 2005

PMID: 15601687

DOI: 10.1128/JB.187.1.45-53.2005


Open Access

Generalized resistance to thymic deletion in the NOD mouse; a polygenic trait characterized by defective induction of Bim.
Liston A, Lesage S, Gray DH, O'Reilly LA, Strasser A, Fahrer AM, Boyd RL, Wilson J, Baxter AG, Gallo EM, Crabtree GR, Peng K, Wilson SR, Goodnow CC

The cause of common polygenic autoimmune diseases is not understood because of genetic and cellular complexity. Here, we pinpoint the action of a subset of autoimmune susceptibility loci in the NOD mouse strain linked to D1mit181, D2mit490, D7mit101, and D15mit229, which cause a generalized resistance to thymic deletion in vivo that applies equally to Aire-induced organ-specific gene products in the thymic medulla and to systemic antigens expressed at high levels throughout the thymus and affects CD4(+), CD4(+)8(+), and CD4(+)25(+) thymocytes. Resistance to thymic deletion does not reflect a general deficit in TCR signaling to calcineurin- or ERK-induced genes, imbalance in constitutive regulators of apoptosis, nor excessive signaling to prosurvival genes but is distinguished by failure to induce the proapoptotic gene and protein, Bim, during in vivo encounter with high-avidity autoantigen. These findings establish defects in thymic deletion and Bim induction as a key mechanism in the pathogenesis of autoimmunity.

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Immunity, 21, 1074-7613, 817-30, 2004

PMID: 15589170


Open Access

Transduction of naive CD4 T cells with kinase-deficient Lck-HIV-Tat fusion protein dampens T cell activation and provokes a switch to regulatory function.
M Veldhoen, AI Magee, MN Penha-Goncalves, B Stockinger

We show here that T cell differentiation can be altered by exposing naive mouse CD4 T cells to altered T cell receptor signaling, achieved by transducing them with a fusion protein consisting of a modified Lck protein lacking the kinase domain and the HIV-Tat protein transduction domain. The Lck-HIV-Tat fusion protein is internalized into naive mouse T cells within 30 min after application to the medium. Activation of transduced cells in vitro resulted in strongly reduced intracellular calcium mobilization, alterations in cytokine profile, and sustained up-regulation of CD25. The cells had suppressive activity in vitro, but no Foxp3 expression. Our data indicate that signals encountered by a naive T cell during its initial activation can profoundly influence its subsequent functional behavior and elicit T cells, which can have regulatory activity.

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European journal of immunology, 35, 1, 207-16, 2005

PMID: 15580657

DOI: 10.1002/eji.200425542


The Corfu deltabeta thalassemia deletion disrupts gamma-globin gene silencing and reveals post-transcriptional regulation of HbF expression.
L Chakalova, CS Osborne, YF Dai, B Goyenechea, A Metaxotou-Mavromati, A Kattamis, C Kattamis, P Fraser

The 7.2 kilobase (kb) Corfu deltabeta thalassemia mutation is the smallest known deletion encompassing a region upstream of the human delta gene that has been suggested to account for the vastly different phenotypes in hereditary persistence of fetal hemoglobin (HPFH) versus beta thalassemia. Fetal hemoglobin (HbF) expression in Corfu heterozygotes and homozygotes is paradoxically dissimilar, suggesting conflicting theories as to the function of the region on globin gene regulation. Here, we measure gamma- and beta-globin gene transcription, steady-state mRNA, and hemoglobin expression levels in primary erythroid cells cultured from several patients with Corfu deltabeta thalassemia. We show through RNA fluorescence in situ hybridization that the Corfu deletion results in high-level transcription of the fetal gamma genes in cis with a concomitant reduction in transcription of the downstream beta gene. Surprisingly, we find that elevated gamma gene transcription does not always result in a corresponding accumulation of gamma mRNA or fetal hemoglobin, indicating a post-transcriptional regulation of gamma gene expression. The data suggest that efficient gamma mRNA accumulation and HbF expression are blocked until beta mRNA levels fall below a critical threshold. These results explain the Corfu paradox and show that the deleted region harbors a critical element that functions in the developmentally regulated transcription of the beta-globin genes.

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Blood, 105, 5, 2154-60, 2005

PMID: 15536151

DOI: 10.1182/blood-2003-11-4069


Open Access

Cell signalling: IP3 receptors channel calcium into cell death.
CJ Hanson, MD Bootman, HL Roderick

There is substantial evidence that Ca2+ fluxes occur during most forms of apoptosis, and that inhibiting such fluxes protects cells from death. IP3 receptors--ligand-gated channels that release Ca2+ from intracellular stores--are emerging as key sites for regulation by pro- and anti-apoptotic factors.

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Current biology : CB, 14, 21, R933-5, 2004

PMID: 15530388

DOI: 10.1016/j.cub.2004.10.019


Resourceful imprinting.
Constância M, Kelsey G, Reik W

Nature, 432, 1476-4687, 53-7, 2004

PMID: 15525980


The Molecular Pages of the mesotelencephalic dopamine consortium (DopaNet).
Le Novère N, Donizelli M

DopaNet http://www.dopanet.org is a Systems Biology initiative that aims to investigate precisely and quantitatively all the aspects of neurotransmission in a specific neuronal system, the mesotelencephalic dopamine system. The project should lead to large-scale models of molecular and cellular processes involved in neuronal signaling. A prerequisite is the proper storage of knowledge coming from the literature.

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BMC bioinformatics, 5, 1471-2105, 174, 2004

PMID: 15518589


Open Access

Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation.
Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, Walter J, Higgins M, Feil R, Reik W

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

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Nature genetics, 36, 1061-4036, 1291-5, 2004

PMID: 15516931


Redox regulation of phosphatase function.
Leslie NR, Lindsay Y, Ross SH, Downes CP

Although reactive oxygen species play important roles in cellular physiology as signalling molecules, their molecular targets are largely unknown. A probable group of targets for mediating many of the effects of reactive oxygen species on cell signalling is the large diverse family of cysteine-dependent phosphatases, which includes the protein tyrosine phosphatases. Our work and that of others suggest that the oxidative inactivation of protein and lipid phosphatases plays an important part in signalling, downstream of many cellular stimuli. Future studies should give us a clearer picture of the role of phosphatase inactivation in cellular behaviour and explain how specificity is achieved in redox signalling.

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Biochemical Society transactions, 32, 0300-5127, 1018-20, 2004

PMID: 15506952


Essential role for the p110delta phosphoinositide 3-kinase in the allergic response.
Ali K, Bilancio A, Thomas M, Pearce W, Gilfillan AM, Tkaczyk C, Kuehn N, Gray A, Giddings J, Peskett E, Fox R, Bruce I, Walker C, Sawyer C, Okkenhaug K, Finan P, Vanhaesebroeck B

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcvarepsilonRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110delta isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen-IgE-induced degranulation and cytokine release. Inactivation of p110delta protects mice against anaphylactic allergic responses. These results identify p110delta as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

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Nature, 431, 1476-4687, 1007-11, 2004

PMID: 15496927


Open Access

Impaired B-1 and B-2 B cell development and atypical splenic B cell structures in IL-7 receptor-deficient mice.
L Erlandsson, S Licence, F Gaspal, S Bell, P Lane, AE Corcoran, IL Mårtensson

The cytokine IL-7 and its receptor are essential for normal B and T lymphopoiesis. We have analyzed the role of this receptor in B cell development throughout ontogeny in IL-7 receptor alpha-deficient mice. We demonstrate that the IL-7 receptor becomes progressively more important with age. B lymphopoiesis takes place, albeit at reduced levels, in fetal liver and bone marrow of young mice, but is arrested in adults. The outcome is a severe reduction, from an early age, in peripheral B cells including follicular, marginal zone and B-1 B cells as well as perturbed splenic B cell structures, which are restored after adoptive transfer of normal spleen cells. We conclude that in the absence of the IL-7 receptor, the residual B lymphopoiesis occurring early in ontogeny must be facilitated by another component, whereas the IL-7 receptor is the key factor in adults. The impairment of marginal zone and B-1 B cells in IL-7 receptor- but not IL-7-deficient mice suggests non-redundant functions for the IL-7 receptor ligands, IL-7 and thymic stromal lymphopoietin.

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European journal of immunology, 34, 12, 3595-603, 2004

PMID: 15495160

DOI: 10.1002/eji.200425217


Gene dosage--limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity.
Liston A, Gray DH, Lesage S, Fletcher AL, Wilson J, Webster KE, Scott HS, Boyd RL, Peltonen L, Goodnow CC

Inactivation of the autoimmune regulator (Aire) gene causes a rare recessive disorder, autoimmune polyendocrine syndrome 1 (APS1), but it is not known if Aire-dependent tolerance mechanisms are susceptible to the quantitative genetic changes thought to underlie more common autoimmune diseases. In mice with a targeted mutation, complete loss of Aire abolished expression of an insulin promoter transgene in thymic epithelium, but had no effect in pancreatic islets or the testes. Loss of one copy of Aire diminished thymic expression of the endogenous insulin gene and the transgene, resulting in a 300% increase in islet-reactive CD4 T cells escaping thymic deletion in T cell receptor transgenic mice, and dramatically increased progression to diabetes. Thymic deletion induced by antigen under control of the thyroglobulin promoter was abolished in Aire homozygotes and less efficient in heterozygotes, providing an explanation for thyroid autoimmunity in APS1. In contrast, Aire deficiency had no effect on thymic deletion to antigen controlled by a systemic H-2K promoter. The sensitivity of Aire-dependent thymic deletion to small reductions in function makes this pathway a prime candidate for more subtle autoimmune quantitative trait loci, and suggests that methods to increase Aire activity would be a potent strategy to lower the incidence of organ-specific autoimmunity.

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The Journal of experimental medicine, 200, 0022-1007, 1015-26, 2004

PMID: 15492124


Open Access

Expression patterns of the novel imprinted genes Nap1l5 and Peg13 and their non-imprinted host genes in the adult mouse brain.
Davies W, Smith RJ, Kelsey G, Wilkinson LS

Recent work has implicated imprinted gene functioning in neurodevelopment and behaviour and defining the expression patterns of these genes in brain tissue has become a key prerequisite to establishing function. In this work we report on the expression patterns of two novel imprinted loci, Nap1l5 and Peg13, in adult mouse brain using in situ hybridisation methods. Nap1l5 and Peg13 are located, respectively, within the introns of the non-imprinted genes Herc3 and the Tularik1 (T1)/KIAA1882 homologue in two separate microimprinted domains on mouse chromosomes 6 and 15. These 'host' genes are highly expressed in brain and consequently we were interested in assessing their expression patterns in parallel to the imprinted genes. The brain expression of all four genes appeared to be mainly neuronal. The detailed expression profiles of Nap1l5 and Peg13 were generally similar with widespread expression that was relatively high in the septal and hypothalamic regions, the hippocampus and the cerebral cortex. In contrast, there was some degree of dissociation between the imprinted genes and their non-imprinted hosts, in that, whilst there was again widespread expression of Herc3 and the T1/KIAA1882 homologue, these genes were also particularly highly expressed in Purkinje neurons and piriform cortex. We also examined expression of the novel imprinted genes in the adrenal glands. Nap1l5 expression was localised mainly to the adrenal medulla, whilst Peg13 expression was observed more generally throughout the adrenal medulla and the outer cortical layers.

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Gene expression patterns : GEP, 4, 1567-133X, 741-7, 2004

PMID: 15465498


Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen.
Pearce AC, Senis YA, Billadeau DD, Turner M, Watson SP, Vigorito E

Vav family proteins are guanine nucleotide exchange factors for the Rho/Rac family of small GTP-binding proteins. In addition, they have domains that mediate protein-protein interactions, including one Src homology 2 (SH2) and two Src homology 3 (SH3) domains. Vav1, Vav2, and Vav3 play a crucial role in the regulation of phospholipase C gamma (PLC gamma) isoforms by immuno-tyrosine-based activation motif (ITAM)-coupled receptors, including the T- and B-cell antigen receptors. We have reported in platelets, however, that Vav1 and Vav2 are not required for activation of PLC gamma 2 in response to stimulation of the ITAM-coupled collagen receptor glycoprotein VI (GPVI). Here we report that Vav3 is tyrosinephosphorylated upon activation of GPVI but that Vav3-deficient platelets also exhibit a normal response upon activation of the ITAM receptor. In sharp contrast, platelets deficient in both Vav1 and Vav3 show a marked inhibition of aggregation and spreading upon activation of GPVI, which is associated with a reduction in tyrosine phosphorylation of PLC gamma 2. The phenotype of Vav1/2/3 triple-deficient platelets is similar to that of Vav1/3 double-deficient cells. These results demonstrate that Vav3 and Vav1 play crucial but redundant roles in the activation of PLC gamma 2 by GPVI. This is the first time that absolute redundancy between two protein isoforms has been observed with respect to the regulation of PLC gamma 2 in platelets.

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The Journal of biological chemistry, 279, 0021-9258, 53955-62, 2004

PMID: 15456756


Open Access

Fetal growth restriction: a workshop report.
Cetin I, Foidart JM, Miozzo M, Raun T, Jansson T, Tsatsaris V, Reik W, Cross J, Hauguel-de-Mouzon S, Illsley N, Kingdom J, Huppertz B

Intrauterine growth restriction (IUGR) is associated with significantly increased perinatal morbidity and mortality as well as cardiovascular disease and glucose intolerance in adult life. A number of disorders from genetic to metabolic, vascular, coagulative, autoimmune, as well as infectious, can influence fetal growth by damaging the placenta, leading to IUGR as a result of many possible fetal, placental and maternal disorders. Strict definitions of IUGR and of its severity are needed in order to eventually distinguish among different phenotypes, such as gestational age at onset, degree of growth restriction and presence of hypoxia. This report explores and reviews some of the most recent developments in both clinical and basic research on intrauterine growth restriction, by seeking mechanisms that involve genetic factors, utero-placental nutrient availability and vascular growth factors. New exciting findings on the genomic imprinting defects potentially associated with IUGR, and the placental anomalies associated with the decreased nutrient transport are summarized. Moreover, recent data on angiogenic growth factors as well as new information arising from application of gene chip technologies are discussed.

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Placenta, 25, 0143-4004, 753-7, 0

PMID: 15450396


Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming.
Morgan HD, Dean W, Coker HA, Reik W, Petersen-Mahrt SK

DNA deaminases of the Aid/Apobec family convert cytosine into uracil and play key roles in acquired and innate immunity. The epigenetic modification by methylation of cytosine in CpG dinucleotides is also mutagenic, but this is thought to occur by spontaneous deamination. Here we show that Aid and Apobec1 are 5-methylcytosine deaminases resulting in a thymine base opposite a guanine. Their action can thus lead to C --> T transition mutations in methylated DNA, or in conjunction with repair of the T:G mismatch, to demethylation. The Aid and Apobec1 genes are located in a cluster of pluripotency genes including Nanog and Stella and are co-expressed with these genes in oocytes, embryonic germ cells, and embryonic stem cells. These results suggest that Aid and perhaps some of its family members may have roles in epigenetic reprogramming and cell plasticity. Transition in CpG dinucleotides is the most frequent mutation in human genetic diseases, and sequence context analysis of CpG transitions in the APC tumor suppressor gene suggests that DNA deaminases may play a significant role in tumor etiology.

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The Journal of biological chemistry, 279, 0021-9258, 52353-60, 2004

PMID: 15448152


Open Access

Active genes dynamically colocalize to shared sites of ongoing transcription.
CS Osborne, L Chakalova, KE Brown, D Carter, A Horton, E Debrand, B Goyenechea, JA Mitchell, S Lopes, W Reik, P Fraser

The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites.

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Nature genetics, 36, 10, 1065-71, 2004

PMID: 15361872

DOI: 10.1038/ng1423


Open Access

Early intermediates of mariner transposition: catalysis without synapsis of the transposon ends suggests a novel architecture of the synaptic complex.
K Lipkow, N Buisine, DJ Lampe, R Chalmers

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.

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Molecular and cellular biology, 24, 18, 8301-11, 2004

PMID: 15340089

DOI: 10.1128/MCB.24.18.8301-8311.2004


Open Access

Promiscuous target interactions in the mariner transposon Himar1.
K Lipkow, N Buisine, R Chalmers

We have previously characterized the early intermediates of mariner transposition. Here we characterize the target interactions that occur later in the reaction. We find that, in contrast to the early transposition intermediates, the strand transfer complex is extremely stable and difficult to disassemble. Transposase is tightly bound to the transposon ends constraining rotation of the DNA at the single strand gaps in the target site flanking the element on either side. We also find that although the cleavage step requires Mg2+ or Mn2+ as cofactor, the strand transfer step is also supported by Ca2+, suggesting that the structure of the active site changes between cleavage and insertion. Finally, we show that, in contrast to the bacterial cut and paste transposons, mariner target interactions are promiscuous and can take place either before or after cleavage of the flanking DNA. This is similar to the behavior of the V(D)J system, which is believed to be derived from an ancestral eukaryotic transposon. We discuss the implications of promiscuous target interactions for promoting local transposition and whether this is an adaptation to facilitate the invasion of a genome following horizontal transfer to a new host species.

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The Journal of biological chemistry, 279, 47, 48569-75, 2004

PMID: 15333635

DOI: 10.1074/jbc.M408759200


Open Access

Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus.
Vigorito E, Bardi G, Glassford J, Lam EW, Clayton E, Turner M

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca(2+) flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca(2+) flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca(2+) flux has a PI3K-independent component, while Ca(2+) flux following mIg/CD19 coligation is totally PI3K dependent. The p85alpha and p110delta subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca(2+) flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110alpha becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.

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Journal of immunology (Baltimore, Md. : 1950), 173, 0022-1767, 3209-14, 2004

PMID: 15322182


Open Access