36 research outputs found
Isolation of Obligate Anaerobic Bacteria from Bovine Abscesses in Sites Other Than the Liver
Comparison of extracellular enzymes of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme
PTEN redundancy: overexpressing lpten, a homolog of Dictyostelium discoideum ptenA, the ortholog of human PTEN, rescues all behavioral defects of the mutant ptenA-.
Mutations in the tumor suppressor gene PTEN are associated with a significant proportion of human cancers. Because the human genome also contains several homologs of PTEN, we considered the hypothesis that if a homolog, functionally redundant with PTEN, can be overexpressed, it may rescue the defects of a PTEN mutant. We have performed an initial test of this hypothesis in the model system Dictyostelium discoideum, which contains an ortholog of human PTEN, ptenA. Deletion of ptenA results in defects in motility, chemotaxis, aggregation and multicellular morphogenesis. D. discoideum also contains lpten, a newly discovered homolog of ptenA. Overexpressing lpten completely rescues all developmental and behavioral defects of the D. discoideum mutant ptenA-. This hypothesis must now be tested in human cells
<i>lpten<sup>−</sup></i> cells translocating in buffer in the absence of chemoattractant exhibit defects in velocity, turning and the suppression of lateral pseudopod formation.
<p>Cells were analyzed in a perfusion chamber through which buffer without attractant was pumped. A. 2D motility parameters of Ax2, <i>lpten<sup>−</sup></i> and <i>ptenA<sup>−</sup>/lpten<sup>oe</sup></i> cells assessed with 2D-DIAS software. B, C, D. 2D-DIAS reconstructions of cell perimeters to generate tracks. Arrows denote net direction, and the blue-filled perimeters represent the last cell positions in the tracks. E, F. 3D-DIAS reconstructions at 0° (top view) and 90° (side view) of representative Ax2 and <i>lpten<sup>−</sup></i> cells, respectively, denoting pseudopods (red). Note that the multiple lateral pseudopods formed by <i>lpten<sup>−</sup></i> cells, were primarily off the substrate. a, anterior end of cell; p, posterior end of cell; lps, lateral pseudopod. G. 2D analysis of lateral pseudopod formation. Inst. vel., instantaneous velocity; No. turns per 10 min., number of turns per 10 minutes; Percent mot. cells, percent motile cells. Parameters are presented as the means ± standard deviations. T-test was used to determine p values. Parameters are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s002" target="_blank">Table S2</a>.</p
Overexpressing <i>lpten<sup>−</sup></i> in the <i>ptenA<sup>−</sup></i> mutant.
<p>A. The transformation vector used to generate strains <i>ptenA<sup>−</sup>/lpten<sup>oe</sup></i>, in which <i>lpten</i> is under the regulation of the <i>actin 15</i> (<i>act15</i>) promoter, fused in frame at the 3′ end to the red fluorescent protein gene (<i>rfp</i>) and terminating with a 3′ <i>actin 8</i> gene sequence. The positions of the primers P8 and P9, for generating the <i>lpten-rfp</i> cDNA, are denoted. Insert shows verification of the <i>lpten-rfp</i> cDNA by PCR. B. <i>lpten</i> is expressed in <i>ptenA<sup>−</sup>/lpten<sup>oe</sup></i> cells at levels more than 10 times that in the parent <i>ptenA<sup>−</sup></i> mutant. The positions of the primers (P1, P2) for RT-PCR of the 300 bp <i>lpten</i> fragment (F) are denoted. In the insert to the right of panel B, RT-PCR products of chemotactically responsive <i>ptenA<sup>−</sup></i> and <i>ptenA<sup>−</sup>/lpten<sup>oe</sup></i> cells reveals overexpression of <i>lpten</i> in the latter. Densitometry measurements revealed>10 fold overexpression. C. Fruiting body formation in Ax2 cultures. D. The absence of fruiting body formation in <i>ptenA<sup>−</sup></i> cultures. E. Fruiting body formation in <i>ptenA<sup>−</sup>/lpten<sup>oe</sup></i> cultures. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s001" target="_blank">Table S1</a> for description of primers.</p