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  • 1
    Language: English
    In: The Journal of cell biology, 05 December 2016, Vol.215(5), pp.735-747
    Description: During epithelial-to-mesenchymal transitions (EMTs), cells disassemble cadherin-based junctions to segregate from the epithelia. Chick premigratory cranial neural crest cells reduce Cadherin-6B (Cad6B) levels through several mechanisms, including proteolysis, to permit their EMT and migration. Serial processing of Cad6B by a disintegrin and metalloproteinase (ADAM) proteins and γ-secretase generates intracellular C-terminal fragments (CTF2s) that could acquire additional functions. Here we report that Cad6B CTF2 possesses a novel pro-EMT role by up-regulating EMT effector genes in vivo. After proteolysis, CTF2 remains associated with β-catenin, which stabilizes and redistributes both proteins to the cytosol and nucleus, leading to up-regulation of β-catenin, CyclinD1, Snail2, and Snail2 promoter-based GFP expression in vivo. A CTF2 β-catenin-binding mutant, however, fails to alter gene expression, indicating that CTF2 modulates β-catenin-responsive EMT effector genes. Notably, CTF2 association with the endogenous Snail2 promoter in the neural crest is β-catenin dependent. Collectively, our data reveal how Cad6B proteolysis orchestrates multiple pro-EMT regulatory inputs, including CTF2-mediated up-regulation of the Cad6B repressor Snail2, to ensure proper cranial neural crest EMT.
    Keywords: Gene Expression Regulation, Developmental ; Proteolysis ; Transcription, Genetic ; Avian Proteins -- Metabolism ; Cadherins -- Metabolism ; Epithelial-Mesenchymal Transition -- Genetics ; Neural Crest -- Cytology
    ISSN: 00219525
    E-ISSN: 1540-8140
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  • 2
    Language: English
    In: Chemosphere, March 2016, Vol.147, pp.239-247
    Description: Bioremediation strategies, including bioaugmentation with chlorinated ethene-degrading enrichment cultures, have been successfully applied in the cleanup of subsurface environments contaminated with tetrachloroethene (PCE) and/or trichloroethene (TCE). However, these compounds are frequently found in the environment as components of mixtures that may also contain chlorinated ethanes and methanes. Under these conditions, the implementation of bioremediation may be complicated by inhibition effects, particularly when multiple dehalorespirers are present. We investigated the ability of the 1,1,2,2-tetrachloroethane (TeCA)-dechlorinating culture WBC-2 to biotransform TeCA alone, or a mixture of TeCA plus PCE and carbon tetrachloride (CT), in microcosms. The microcosms contained electron donors provided to biostimulate the added culture and sediment collected from a wetland where numerous “hotspots” of contamination with chlorinated solvent mixtures exist. The dominant TeCA biodegradation mechanism mediated by the WBC-2 culture in the microcosms was different in the presence of these wetland sediments than in the sediment-free enrichment culture or in previous WBC-2 bioaugmented microcosms and column tests conducted with wetland sediment collected at nearby sites. The co-contaminants and their daughter products also inhibited TeCA biodegradation by WBC-2. These results highlight the need to conduct biodegradability assays at new sites, particularly when multiple contaminants and dehalorespiring populations are present.
    Keywords: 1,1,2,2-Tetrachloroethane ; Carbon Tetrachloride ; Tetrachloroethene ; Contaminated Wetlands ; Dehalococcoides ; Dehalobacter ; Chemistry ; Ecology
    ISSN: 0045-6535
    E-ISSN: 1879-1298
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  • 3
    In: Molecular Reproduction and Development, October 2013, Vol.80(10), pp.826-839
    Description: Byline: Andrew T. Schiffmacher, Carol L. Keefer SUMMARY The bovine trophectoderm (TE) undergoes a dramatic morphogenetic transition prior to uterine endometrial attachment. Many studies have documented trophoblast-specific gene expression profiles at various pre-attachment stages, yet genetic interactions within the transitioning TE gene regulatory network are not well characterized. During bovine embryogenesis, transcription factors OCT4 and CDX2 are co-expressed during early trophoblast elongation. In this study, the bovine trophectoderm-derived CT-1 cell line was utilized as a genetic model to examine the roles of CDX2 and OCT4 within the bovine trophoblast gene regulatory network. An RT-PCR screen for TE-lineage transcription factors identified expression of CDX2, ERRB, ID2, SOX15, ELF5, HAND1, and ASCL2. CT-1 cells also express a nuclear-localized, 360 amino acid OCT4 ortholog of the pluripotency-specific human OCT4A. To delineate the roles of CDX2 and OCT4 within the CT-1 gene network, CDX2 and OCT4 levels were manipulated via overexpression and siRNA-mediated knockdown. An increase in CDX2 negatively regulated OCT4 expression, but increased expression of IFNT, HAND1, ASCL2, SOX15, and ELF5. A reduction of CDX2 levels exhibited a reciprocal effect, resulting in decreased expression of IFNT, HAND1, ASCL2, and SOX15. Both overexpression and knockdown of CDX2 increased ETS2 transcription. In contrast to CDX2, manipulation of OCT4 levels only revealed a positive autoregulatory mechanism and upregulation of ASCL2. Together, these results suggest that CDX2 is a core regulator of multiple trophoblast genes within CT-1 cells. Mol. Reprod. Dev. 80: 826-839, 2013. [c] 2013 Wiley Periodicals, Inc. Supporting information: Additional Supporting Information may be found in the online version of this article Additional supporting information may be found in the online version of this article at the publisher's web-site. CAPTION(S): Figure S1. Expression profile of selected genes in the CT-1 cell line that are also expressed during bovine or mouse trophoblast development. Three replicates of cDNAs from CT-1 cells grown on a collagen substrate or substrate-free CellBIND[R] were tested. cDNAs derived from CT-1 cells cultured on STO mouse embryonic fibroblasts or STO cells alone were analyzed as controls for STO cDNA contamination. cDNAs from mid-gestation cotyledon and pooled IVP blastocysts served as positive controls where appropriate. Figure S2. siRNA mediated downregulation of OCT4 reduces endogenous OCT4 levels, but does not significantly regulate gene expression in a subset of bovine trophoblast-specific genes. Data are represented as the siRNA treatment means and standard errors of normalized Ct values relative to the ACTG1-normalized, non-transfected-control treatment Ct mean (set at 1) for each gene. Means are generated from 3 independent experimental cDNA replicates. Asterisks (*) located above values denote a significant difference between that treatment value and all other values of the corresponding gene (P 0.05). Black bars indicate nontransfected control levels, dark gray bars indicate mis-match siRNA-transfected control levels, and light gray bars indicate OCT4 siRNA-transfected levels. Figure S3. mRNA levels of transcription factors CDX2 (A) and OCT4 (B) following siRNA knockdown in CT-1 cells. Two siRNA concentrations (50 vs. 100nM) were tested at two Lipitoid/DNA ([+ or -]) charge ratios (3/1 vs. 4/1). For each treatment, a non-specific control siRNA was also transfected to serve as a negative control. Each transfection treatment is expressed as the mean and standard error (n=3) of the siRNA target relative expression level normalized to treatment control levels. An asterisk (*) denotes a significant difference between treatment effect and untransfected control (P 0.05).
    Keywords: Transcription (Genetics) -- Analysis ; Genetic Research -- Analysis ; Genes -- Analysis;
    ISSN: 1040-452X
    E-ISSN: 1098-2795
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  • 4
    Language: English
    In: Developmental Biology, 01 December 2018, Vol.444, pp.S237-S251
    Description: During epithelial-to-mesenchymal transitions (EMTs), chick cranial neural crest cells simultaneously delaminate from the basement membrane and segregate from the epithelia, in part, via multiple protease-mediated mechanisms. Proteolytic processing of Cadherin-6B (Cad6B) in premigratory cranial neural crest cells by metalloproteinases not only disassembles cadherin-based junctions but also generates shed Cad6B ectodomains or N-terminal fragments (NTFs) that may possess additional roles. Here we report that Cad6B NTFs promote delamination by enhancing local extracellular proteolytic activity around neural crest cells undergoing EMT . During EMT, Cad6B NTFs of varying molecular weights are observed, indicating that Cad6B may be cleaved at different sites by A Disintegrin and Metalloproteinases (ADAMs) 10 and 19 as well as by other matrix metalloproteinases (MMPs). To investigate Cad6B NTF function, we first generated NTF constructs that express recombinant NTFs with similar relative mobilities to those NTFs shed . Overexpression of either long or short Cad6B NTFs in premigratory neural crest cells reduces laminin and fibronectin levels within the basement membrane, which then facilitates precocious neural crest cell delamination. Zymography assays performed with supernatants of neural crest cell explants overexpressing Cad6B long NTFs demonstrate increased MMP2 activity versus controls, suggesting that Cad6B NTFs promote delamination through a mechanism involving MMP2. Interestingly, this increase in MMP2 does not involve up-regulation of or its regulators at the transcriptional level but instead may be attributed to a physical interaction between shed Cad6B NTFs and MMP2. Taken together, these results highlight a new function for Cad6B NTFs and provide insight into how cadherins regulate cellular delamination during normal developmental EMTs as well as aberrant EMTs that underlie human disease.
    Keywords: Biology ; Zoology
    ISSN: 0012-1606
    E-ISSN: 1095-564X
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  • 5
    Language: English
    In: Developmental Biology, 01 May 2017, Vol.425(1), pp.85-99
    Description: Cranial sensory ganglia are components of the peripheral nervous system that possess a significant somatosensory role and include neurons within the trigeminal and epibranchial nerve bundles. Although it is well established that these ganglia arise from interactions between neural crest and neurogenic placode cells, the molecular basis of ganglia assembly is still poorly understood. Members of the Annexin protein superfamily play key roles in sensory nervous system development throughout metazoans. Annexin A6 is expressed in chick trigeminal and epibranchial placode cell-derived neuroblasts and neurons, but its function in cranial ganglia formation has not been elucidated. To this end, we interrogated the role of Annexin A6 using gene perturbation studies in the chick embryo. Our data reveal that placode cell-derived neuroblasts with reduced Annexin A6 levels ingress and migrate normally to the ganglionic anlage, where neural crest cell corridors correctly form around them. Strikingly, while Annexin A6-depleted placode cell-derived neurons still express mature neuronal markers, they fail to form two long processes, which are considered morphological features of mature neurons, and no longer innervate their designated targets due to the absence of this bipolar morphology. Moreover, overexpression of Annexin A6 causes some placode cell-derived neurons to form extra protrusions alongside these bipolar processes. These data demonstrate that the molecular program associated with neuronal maturation is distinct from that orchestrating changes in neuronal morphology, and, importantly, reveal Annexin A6 to be a key membrane scaffolding protein during sensory neuron membrane biogenesis. Collectively, our results provide novel insight into mechanisms underscoring morphological changes within placode cell-derived neurons that are essential for cranial gangliogenesis.
    Keywords: Cranial Ganglia ; Placode Cells ; Neural Crest Cells ; Annexin A6 ; Membrane Dynamics ; Neurons ; Biology ; Zoology
    ISSN: 0012-1606
    E-ISSN: 1095-564X
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  • 6
    Language: English
    In: CELLULAR REPROGRAMMING, August 2011, Vol.13(4)
    Description: Atomic force microscopy (AFM) has emerged as a promising tool to characterize the mechanical properties of biological materials and cells. In our studies, undifferentiated and early differentiating mouse embryonic stem cells (mESCs) were assessed individually using an AFM system to determine...
    Keywords: Engineering Sciences ; Automatic
    ISSN: 21524971
    E-ISSN: 21524998
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  • 7
    Language: English
    In: In Vitro Cellular & Developmental Biology - Animal, 2012, Vol.48(7), pp.403-406
    Description: Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.
    Keywords: Bovine ; CT-1 ; Lipitoid ; Transfection ; Trophectoderm
    ISSN: 1071-2690
    E-ISSN: 1543-706X
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  • 8
    Language: English
    In: Progress in Molecular Biology and Translational Science: The Molecular Biology of Cadherins
    Keywords: Proteins
    ISBN: 978-0-12-394647-8
    Source: Gale Virtual Reference Library (GVRL)
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  • 9
    Language: English
    In: Molecular biology of the cell, January 2014, Vol.25(1), pp.41-54
    Description: The epithelial-to-mesenchymal transition (EMT) is a highly coordinated process underlying both development and disease. Premigratory neural crest cells undergo EMT, migrate away from the neural tube, and differentiate into diverse cell types during vertebrate embryogenesis. Adherens junction disassembly within premigratory neural crest cells is one component of EMT and, in chick cranial neural crest cells, involves cadherin-6B (Cad6B) down-regulation. Whereas Cad6B transcription is repressed by Snail2, the rapid loss of Cad6B protein during EMT is suggestive of posttranslational mechanisms that promote Cad6B turnover. For the first time in vivo, we demonstrate Cad6B proteolysis during neural crest cell EMT, which generates a Cad6B N-terminal fragment (NTF) and two C-terminal fragments (CTF1/2). Coexpression of relevant proteases with Cad6B in vitro shows that a disintegrin and metalloproteinases (ADAMs) ADAM10 and ADAM19, together with γ-secretase, cleave Cad6B to produce the NTF and CTFs previously observed in vivo. Of importance, both ADAMs and γ-secretase are expressed in the appropriate spatiotemporal pattern in vivo to proteolytically process Cad6B. Overexpression or depletion of either ADAM within premigratory neural crest cells prematurely reduces or maintains Cad6B, respectively. Collectively these results suggest a dual mechanism for Cad6B proteolysis involving two ADAMs, along with γ-secretase, during cranial neural crest cell EMT.
    Keywords: Epithelial-Mesenchymal Transition ; Avian Proteins -- Metabolism ; Cadherins -- Metabolism ; Neural Crest -- Cytology ; Skull -- Embryology
    ISSN: 10591524
    E-ISSN: 1939-4586
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  • 10
    Language: English
    In: The Journal of infectious diseases, 01 July 2003, Vol.188(1), pp.114-7
    Description: An adult with chronic obstructive pulmonary disease was monitored prospectively for 2 years. Nontypeable Haemophilus influenzae was isolated from sputum cultures at 22 of 23 monthly clinic visits. Analysis of the isolates, by pulsed-field gel electrophoresis (PFGE), revealed that the patient was colonized by 3 different strains during the 2-year period. The gene encoding outer-membrane protein (OMP) P2, ompP2, was amplified from sputum samples and selected strains obtained from this patient. Analysis of the ompP2 sequences, in combination with the PFGE patterns, indicated that ompP2 horizontal transfer between 2 strains occurred in the respiratory tract, between clinic visits 13 and 14. Observation of ompP2 horizontal transfer in the human respiratory tract has important implications for both the understanding of ompP2 diversity among strains and the future design of OMP P2-based vaccines.
    Keywords: Bacterial Outer Membrane Proteins -- Genetics ; Gene Transfer, Horizontal -- Genetics ; Haemophilus Infections -- Complications ; Haemophilus Influenzae -- Genetics ; Pulmonary Disease, Chronic Obstructive -- Complications
    ISSN: 0022-1899
    E-ISSN: 15376613
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