Some studies on lipids and lipase activities in Streptomyces rimosus

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A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q- syndrome.

The identification of the genes associated with chromosomal translocation breakpoints has fundamentally changed understanding of the molecular basis of hematological malignancies. By contrast, the study of chromosomal deletions has been hampered by the large number of genes deleted and the complexity of their analysis. We report the generation of a mouse model for human 5q- syndrome using large-scale chromosomal engineering. Haploinsufficiency of the Cd74-Nid67 interval (containing Rps14, encoding the ribosomal protein S14) caused macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow. These effects were associated with defective bone marrow progenitor development, the appearance of bone marrow cells expressing high amounts of the tumor suppressor p53 and increased bone marrow cell apoptosis. Notably, intercrossing with p53-deficient mice completely rescued the progenitor cell defect, restoring common myeloid progenitor and megakaryocytic-erythroid progenitor, granulocyte-monocyte progenitor and hematopoietic stem cell bone marrow populations. This mouse model suggests that a p53-dependent mechanism underlies the pathophysiology of the 5q- syndrome

The Ews-ERG Fusion Protein Can Initiate Neoplasia from Lineage-Committed Haematopoietic Cells

The EWS-ERG fusion protein is found in human sarcomas with the chromosomal translocation t(21;22)(q22;q12), where the translocation is considered to be an initiating event in sarcoma formation within uncommitted mesenchymal cells, probably long-lived progenitors capable of self renewal. The fusion protein may not therefore have an oncogenic capability beyond these progenitors. To assess whether EWS-ERG can be a tumour initiator in cells other than mesenchymal cells, we have analysed Ews-ERG fusion protein function in a cellular environment not typical of that found in human cancers, namely, committed lymphoid cells. We have used Ews-ERG invertor mice having an inverted ERG cDNA cassette flanked by loxP sites knocked in the Ews intron 8, crossed with mice expressing Cre recombinase under the control of the Rag1 gene to give conditional, lymphoid-specific expression of the fusion protein. Clonal T cell neoplasias arose in these mice. This conditional Ews gene fusion model of tumourigenesis shows that Ews-ERG can cause haematopoietic tumours and the precursor cells are committed cells. Thus, Ews-ERG can function in cells that do not have to be pluripotent progenitors or mesenchymal cells

Group-2 innate lymphoid cell-dependent regulation of tissue neutrophil migration by alternatively activated macrophage-secreted Ear11.

Type-2 immunity is characterised by interleukin (IL)-4, IL-5 and IL-13, eosinophilia, mucus production, IgE, and alternatively activated macrophages (AAM). However, despite the lack of neutrophil chemoattractants such as CXCL1, neutrophils, a feature of type-1 immunity, are observed in type-2 responses. Consequently, alternative mechanisms must exist to ensure that neutrophils can contribute to type-2 immune reactions without escalation of deleterious inflammation. We now demonstrate that type-2 immune-associated neutrophil infiltration is regulated by the mouse RNase A homologue, eosinophil-associated ribonuclease 11 (Ear11), which is secreted by AAM downstream of IL-25-stimulated ILC2. Transgenic overexpression of Ear11 resulted in tissue neutrophilia, whereas Ear11-deficient mice have fewer resting tissue neutrophils, whilst other type-2 immune responses are not impaired. Notably, administration of recombinant mouse Ear11 increases neutrophil motility and recruitment. Thus, Ear11 helps maintain tissue neutrophils at homoeostasis and during type-2 reactions when chemokine-producing classically activated macrophages are infrequently elicited

Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment

Assembly of transgenic human P301S Tau is necessary for neurodegeneration in murine spinal cord

Abstract A pathological pathway leading from soluble monomeric to insoluble filamentous Tau is characteristic of many human neurodegenerative diseases, which also exhibit dysfunction and death of brain cells. However, it is unknown how the assembly of Tau into filaments relates to cell loss. To study this, we first used a mouse line transgenic for full-length human mutant P301S Tau to investigate the temporal relationship between Tau assembly into filaments, assessed using anti-Tau antibody AT100, and motor neuron numbers, in the lumbar spinal cord. AT100 immunoreactivity preceded nerve cell loss. Murine Tau did not contribute significantly to either Tau aggregation or neurodegeneration. To further study the relevance of filament formation for neurodegeneration, we deleted hexapeptides 275VQIINK280 and 306VQIVYK311, either singly or in combination, from human 0N4R Tau with the P301S mutation. These hexapeptides are essential for the assembly of Tau into filaments. Homozygous mice transgenic for P301S Tau with the hexapeptide deletions, which expressed Tau at a similar level to the heterozygous line transgenic for P301S Tau, had a normal lifespan, unlike mice from the P301S Tau line. The latter had significant levels of sarkosyl-insoluble Tau in brain and spinal cord, and exhibited neurodegeneration. Mice transgenic for P301S Tau with the hexapeptide deletions failed to show significant levels of sarkosyl-insoluble Tau or neurodegeneration. Recombinant P301S Tau with the hexapeptide deletions failed to form β-sheet structure and filaments following incubation with heparin. Taken together, we conclude that β-sheet assembly of human P301S Tau is necessary for neurodegeneration in transgenic mice

Clonality of T Cell Neoplasias in<i>Ews-ERG</i> Invertor Mice

<div><p>Genomic DNA was prepared from various tissues of invertor mice with thymomas. Genomic analysis was carried out using filter hybridisation to assess the presence of the inverted <i>ERG</i> cassette in the tumour cells (A), and to assess whether the lymphocytes involved in the tumours were clonal T (B) or B cells (C).</p> <p>(A) Inversion hybridisation autoradiograph. DNA was prepared from the thymoma and other tissues of M18, cleaved with EcoRI and hybridised to the 5′ <i>Ews</i> probe (which detects a 5-kb targeted <i>ERG</i> cassette fragment or a 6.5-kb Cre-inverted fragment). If the <i>ERG</i> cassette is inverted by Cre activity, the size of the EcoRI fragment increases from the initial targeted gene size, as indicated in the maps below the figure. The data shown are for DNA extracted from spleen (spl), thymus (thy), liver (liv), kidney (kid) or tail (ES cell DNA is used as a control). The Cre-mediated inverted band (∼6.5 kb) is evident in thymus DNA (thymoma). The summary of the data from the cohort of invertor mice is in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-t002" target="_blank">Table 2</a>. Note that despite extensive infiltration of spleen detected by histology, we cannot see the inverted band by Southern blot; this presumably reflects regional clustering of neoplastic cells in spleen. Restriction fragment sizes are represented as germ line (GL), inverted allele of <i>ERG</i> cassette (inv) and initial targeted <i>Ews</i> allele (Tgt). The organisation of the targeted <i>Ews</i> allele is indicated underneath. The hybridisation autoradiograph shows the location of <i>Ews</i> exon 7 and the initial targeted orientation (bottom) or inverted orientation of <i>ERG</i> invertor cassette after Cre-mediated recombination (top). a, acceptor splice site.</p> <p>(B) Autoradiograph showing rearrangement of T cell receptor β locus. A T cell receptor Jβ2 probe [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b40" target="_blank">40</a>] (diagrammatically shown below the autoradiograph) detects a 5-kb germ line HindIII Jβ2 band whilst V-D-Jβ2 joins in T cells result in new HindIII-sized bands depending on the nature of the rearrangement. Each of 12 thymoma DNAs that were compared showed one or two Jβ2 alleles rearranged, signifying that these tumours were clonal T cells. In some thymoma samples, there is almost complete absence of the germ line band, indicating that the thymuses of these mice are solely comprised of malignant clonal T cells. DNA sequence analysis of the V-D-J junctions of mice M2, M5, M13, and M18 showed functional V-D-J joins (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-t002" target="_blank">Table 2</a>).</p> <p>(C) Autoradiograph showing rearrangement status of immunoglobulin heavy-chain genes. A Cμ intron probe [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030242#pbio-0030242-b41" target="_blank">41</a>] (diagrammatically shown below the autoradiograph) was used to hybridise a set of thymoma DNAs for the presence of <i>Igh</i> rearranged bands. Only two samples showed rearrangements.</p> <p>(D) Detection of Ews-Erg fusion protein in thymoma cells. Single cell suspensions were made of T cells from a normal thymus (wt) or from the thymoma of M6, protein fractionated on 4%–20% acrylamide gel, and transferred to nylon membranes. Specific proteins were detected with anti-Ews or anti-ERG antibody.</p></div