, a bio/informatics shared resource is still "open for business" - Visit the CDS website
The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
UNLABELLED - Members of the APOBEC3 family of cytidine deaminases vary in their proportions of a virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization (C. Song, L. Sutton, M. Johnson, R. D'Aquila, J. Donahue, J Biol Chem 287:16965-16974, 2012, http://dx.doi.org/10.1074/jbc.M111.310839). We have now determined an additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Alterations to one of these specific leucine residues in either of the two A3F CD domains (A3F L368A, L122A, and L184A) decreased core localization and diminished HIV restriction without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A or A3F L368A plus L122A) still were packaged into virions but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization.
IMPORTANCE - Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Nkx2.2 encodes a homeodomain transcription factor required for the correct specification and/or differentiation of cells in the pancreas, intestine, and central nervous system (CNS). To follow the fate of cells deleted for Nkx2.2 within these tissues, we generated Nkx2.2:lacZ knockin mice using a recombination-mediated cassette exchange (RMCE) approach. Expression analysis of lacZ and/or β-galactosidase in Nkx2.2(lacZ/+) heterozygote embryos and adults demonstrates that lacZ faithfully recapitulates endogenous Nkx2.2 expression. Furthermore, the Nkx2.2(lacZ/lacZ) homozygous embryos display phenotypes indistinguishable from the previously characterized Nkx2.2(-/-) strain. LacZ expression analyses in the Nkx2.2(lacZ/lacZ) homozygous embryos indicate that Nkx2.2-expressing progenitor cells within the pancreas are generated in their normal numbers and are not mislocalized within the pancreatic ductal epithelium or developing islets. In the CNS of Nkx2.2(lacZ/lacZ) embryos, LacZ-expressing cells within the ventral P3 progenitor domain display different migration properties depending on the developmental stage and their respective differentiation potential.
Copyright © 2012 Wiley Periodicals, Inc.
Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER(Tm) and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1(PB)-CreER(Tm);R26R(lacZ) mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.
Prior studies showed that bone regeneration during distraction osteogenesis (DO) was dependent on vascular tissue development and that inhibition of VEGFR signaling diminished the expression of BMP2. A combination of micro-computed tomography (μCT) analysis of vascular and skeletal tissues, immunohistological and histological analysis of transgenic mice containing a BAC transgene in which β-galactosidase had been inserted into the coding region of BMP2 and qRT-PCR analysis, was used to examine how the spatial temporal expression of the morphogenetic signals that drive skeletal and vascular tissue development is coordinated during DO. These results showed that BMP2 expression was induced in smooth muscle and vascular endothelial cells of arteries and veins, capillary endothelial cells, hypertrophic chondrocytes and osteocytes. BMP2 was not expressed by lymphatic vessels or macrophages. Separate peaks of BMP2 mRNA expression were induced in the surrounding muscular tissues and the distraction gap and corresponded first with large vessel collateralization and arteriole remodeling followed by periods of angiogenesis in the gap region. Immunohistological and qRT-PCR analysis of VEGF receptors and ligands showed that mesenchymal cells, lining cells and chondrocytes, expressed VEGFA, although PlGF expression was only seen in mesenchymal cells within the gap region. On the other hand VEGFR2 appeared to be predominantly expressed by vascular endothelial and hematopoietic cells. These results suggest that bone and vascular tissue formation is coordinated via a mutually supporting set of paracrine loops in which blood vessels primarily synthesize the morphogens that promote bone formation while mesenchymal cells primarily synthesize the morphogens that promote vascular tissue formation.
Copyright © 2012 Elsevier Inc. All rights reserved.
OBJECTIVE - Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5) in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia.
DESIGN - Lgr5-EGFP-IRES-Cre(ERT2/+);Rosa26R mice were used to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L-635 to induce acute SPEM. Lineage mapping was performed to determine if Lgr5-expressing cells gave rise to SPEM.
RESULTS - Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES-Cre(ERT2/+);Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed.
CONCLUSION - The results indicate that, while chief cells with Lgr5 transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.
Escherichia coli nucleoside diphosphate (NDP) kinase mutants have an increased frequency of spontaneous mutation, possibly due to uracil misincorporation into DNA. Here we show that NDP kinase mutants are dependent on translesion DNA synthesis, often a mutagenic form of DNA synthesis, to prevent mutagenesis.
The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.
Single-dose intratracheal bleomycin has been instrumental for understanding fibrotic lung remodeling, but fails to recapitulate several features of idiopathic pulmonary fibrosis (IPF). Since IPF is thought to result from recurrent alveolar injury, we aimed to develop a repetitive bleomycin model that results in lung fibrosis with key characteristics of human disease, including alveolar epithelial cell (AEC) hyperplasia. Wild-type and cell fate reporter mice expressing β-galactosidase in cells of lung epithelial lineage were given intratracheal bleomycin after intubation, and lungs were harvested 2 wk after a single or eighth biweekly dose. Lungs were evaluated for fibrosis and collagen content. Bronchoalveolar lavage (BAL) was performed for cell counts. TUNEL staining and immunohistochemistry were performed for pro-surfactant protein C (pro-SP-C), Clara cell 10 (CC-10), β-galactosidase, S100A4, and α-smooth muscle actin. Lungs from repetitive bleomycin mice had marked fibrosis with prominent AEC hyperplasia, similar to usual interstitial pneumonia (UIP). Compared with single dosing, repetitive bleomycin mice had greater fibrosis by scoring, morphometry, and collagen content; increased TUNEL+ AECs; and reduced inflammatory cells in BAL. Sixty-four percent of pro-SP-C+ cells in areas of fibrosis expressed CC-10 in the repetitive model, suggesting expansion of a bronchoalveolar stem cell-like population. In reporter mice, 50% of S100A4+ lung fibroblasts were derived from epithelial mesenchymal transition compared with 33% in the single-dose model. With repetitive bleomycin, fibrotic remodeling persisted 10 wk after the eighth dose. Repetitive intratracheal bleomycin results in marked lung fibrosis with prominent AEC hyperplasia, features reminiscent of UIP.
PURPOSE - Mammalian target of rapamycin (mTOR)-mediated pathways play central roles in regulating aging. The purpose of the present study was to characterize the mTOR cascade in human retinal pigment epithelial (RPE) cells and to investigate its potential roles in controlling RPE senescence.
METHODS - Expression of major components of the mTOR signaling networks was evaluated by Western blot analyses. Formations of the two signaling complexes of mTOR, mTORC1, and mTORC2 were determined by coimmunoprecipitation. The activation of mTORC1 was monitored by measuring the phosphorylation status of the downstream substrate protein S6. Senescence of the cultured human RPE cells was assessed by measuring both the senescence associated-β-galactosidase (SA-β-Gal) activity and the expression level of p16, a cyclin-dependent kinase inhibitor.
RESULTS - Human RPE cells contained functional mTORC1 and mTORC2 signaling complexes. The assembly and activity of mTORC1 were regulated by upstream nutrient and growth factor signals. The sensitivity of mTORC1 to extracellular nutrient stimuli increased in RPE cells that had developed in vitro senescence. Suppression of the mTORC1 by rapamycin prevented the appearance of senescence markers in the RPE.
CONCLUSIONS - The mTOR pathway presented age-associated changes in human RPE cells, and downregulation of mTORC1 could delay the aging process of the RPE.
Several transcriptional activators, called "classical" because each bears a natural acidic activating region attached to a DNA binding domain, are proteolytically unstable in yeast, and it has been suggested that this instability is required for transcriptional activation. Here we test the generality of that proposal by examining a set of activators (called "nonclassical") that lack activating regions. These activators (e.g., LexA-Gal11) comprise a LexA DNA binding domain fused to a component of the Mediator and are believed to insert the latter into the Mediator and recruit it (and, indirectly, other components required for transcription) to a gene bearing LexA sites. We find that three, and only three, Mediator subunits, all from its tail domain, work as activators when fused to LexA. All three are unstable, and for the case analyzed in detail, stabilization decreases activity. Thus, to the extent tested, both classical and nonclassical activators work most efficiently when proteolytically unstable.
(c) 2010 Elsevier Ltd. All rights reserved.