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21.
Chrisna Durandt Fiona A. van Vollenstee Carla Dessels Karlien Kallmeyer Danielle de Villiers Candice Murdoch Marnie Potgieter Michael S. Pepper 《Journal of lipid research》2016,57(4):729-742
The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36intermediate/high during adipocyte differentiation in vitro. The gradual increase of CD36intermediate/high/NRpositive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression. 相似文献
22.
Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids 总被引:1,自引:0,他引:1
Brooks JP Maxwell SL Rensing C Gerba CP Pepper IL 《Canadian journal of microbiology》2007,53(5):616-622
The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities. 相似文献
23.
Cell differentiation in multicellular organisms has the obvious function during development of creating new cell types. However, in long-lived organisms with extensive cell turnover, cell differentiation often continues after new cell types are no longer needed or produced. Here, we address the question of why this is true. It is believed that multicellular organisms could not have arisen or been evolutionarily stable without possessing mechanisms to suppress somatic selection among cells within organisms, which would otherwise disrupt organismal integrity. Here, we propose that one such mechanism is a specific pattern of ongoing cell differentiation commonly found in metazoans with cell turnover, which we call “serial differentiation.” This pattern involves a sequence of differentiation stages, starting with self-renewing somatic stem cells and proceeding through several (non–self-renewing) transient amplifying cell stages before ending with terminally differentiated cells. To test the hypothesis that serial differentiation can suppress somatic evolution, we used an agent-based computer simulation of cell population dynamics and evolution within tissues. The results indicate that, relative to other, simpler patterns, tissues organized into serial differentiation experience lower rates of detrimental cell-level evolution. Self-renewing cell populations are susceptible to somatic evolution, while those that are not self-renewing are not. We find that a mutation disrupting differentiation can create a new self-renewing cell population that is vulnerable to somatic evolution. These results are relevant not only to understanding the evolutionary origins of multicellularity, but also the causes of pathologies such as cancer and senescence in extant metazoans, including humans. 相似文献
24.
Kirk C. Hansen Lauren Kiemele Ori Maller Jenean O'Brien Aarthi Shankar Jaime Fornetti Pepper Schedin 《Molecular & cellular proteomics : MCP》2009,8(7):1648-1657
Epithelial cell behavior is coordinated by the composition of the surrounding
extracellular matrix (ECM); thus ECM protein identification is critical for
understanding normal biology and disease states. Proteomic analyses of ECM
proteins have been hindered by the insoluble and digestion-resistant nature of
ECM. Here we explore the utility of combining rapid ultrasonication- and
surfactant-assisted digestion for the detailed proteomics analysis of ECM
samples. When compared with traditional overnight digestion, this optimized
method dramatically improved the sequence coverage for collagen I, revealed the
presence of hundreds of previously unidentified proteins in Matrigel, and
identified a protein profile for ECM isolated from rat mammary glands that was
substantially different from that found in Matrigel. In a three-dimensional
culture assay to investigate epithelial cell-ECM interactions, mammary
epithelial cells were found to undergo extensive branching morphogenesis when
plated with mammary gland-derived matrix in comparison with Matrigel.
Cumulatively these data highlight the tissue-specific nature of ECM composition
and function and underscore the need for optimized techniques, such as those
described here, for the proteomics characterization of ECM samples.Extracellular matrix (ECM)1 is a critical component of the tissue microenvironment. ECM plays a pivotal role
in embryonic stem cell development and differentiation (1, 2) as well as many physiological (3) and pathological processes, including cancer
progression (4, 5). Cell regulation by ECM has been studied with high frequency in recent years
(7, 8).
However, our ability to globally characterize ECM composition both in
vitro and in vivo has been severely limited because of several
unique attributes of ECM proteins such as high molecular weight glycans and the presence
of covalent protein cross-links (6, 9, 10).
Traditional proteomics approaches have proven to be ineffective for the identification
of ECM proteins as demonstrated by the fact that collagens, despite being the most
abundant protein in mammals, are significantly underrepresented in tissue-based
proteomics data sets.Ultrasonication has long been used for the digestion of bioorganic materials to allow for
maximal and reproducible extraction and hence the accurate identification of small
molecule and inorganic analytes (11). More
recently, Capelo et al. (12)
have used ultrasonic energy to catalyze tryptic digestion of proteins for subsequent
mass spectrometry-based identification. Here we sought to determine whether this method
could be optimized to prepare ECM samples for mass spectrometry-based analysis. For
method development, we used rat tail collagen as a representative ECM protein for which
current proteomics approaches have proven relatively unsuccessful. Type I collagen is
defined as a right-handed triple helix heterotrimer comprising two identical
α1 chains and one α2 chain that form a fibrillar network (6). The physical properties of the triple helical
structure render the protein resistant to proteasch as trypsin (9). In this work, we focused our efforts on developing a digestion
approach that improves our ability to perform proteomics analysis on a type I collagen
preparation and then used this method to identify the protein composition of EHS murine
chondrosarcoma matrix (10), herein referred to as
Matrigel, and a matrix preparation from rat mammary tissue.In this study, we developed a digestion approach suitable for a two-dimensional liquid
chromatography-tandem mass spectrometry-based analysis of ECM proteins. Our digestion
approach involves three cycles of ultrasonication for rapid initial trypsin digestion
followed by overnight digestion using an acid-labile surfactant. This approach resulted
in significant improvement in collagen peptide identification and the identification of
numerous ECM proteins previously uncharacterized in Matrigel and in mammary tissue. The
application of our ECM-optimized ultrasonic assisted trypsin digestion method is
anticipated to significantly advance the identification of tissue- and disease
state-specific ECM proteins. 相似文献
25.
Driscoll WW Pepper JW Pierson LS Pierson EA 《Applied and environmental microbiology》2011,77(20):7227-7235
Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are "cheaters" that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions. 相似文献
26.
Miles SL Sinclair RG Riley MR Pepper IL 《Applied and environmental microbiology》2011,77(8):2813-2816
This study evaluated real-time sensing of Escherichia coli as a microbial contaminant in water distribution systems. Most sensors responded to increased E. coli concentrations, showing that select sensors can detect microbial water quality changes and be utilized as part of a contaminant warning system. 相似文献
27.
Brockmeyer C Paster W Pepper D Tan CP Trudgian DC McGowan S Fu G Gascoigne NR Acuto O Salek M 《The Journal of biological chemistry》2011,286(9):7535-7547
Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive "signaling waves" revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function. 相似文献
28.
Pepper JW 《Evolution; international journal of organic evolution》2008,62(12):3185-3191
Many of the greatest challenges in medicine and public health involve the evolution of drug resistance by pathogens. Recent advances in the theory of natural selection suggest that there are two broad classes of pathogen traits that can be targeted by drugs or vaccines. The first class, consisting of traits that benefit the individual organisms bearing them, causes a strong evolutionary response and the rapid emergence of drug resistance. The second class, consisting of traits that benefit groups of pathogen organisms including the individual provider, causes a weaker evolutionary response and less drug resistance. Although most previous drug development has targeted the first class, it would be advantageous to focus on the second class as targets for drug and vaccine development. Specific examples and test cases are discussed. 相似文献
29.
Laffin B Wellberg E Kwak HI Burghardt RC Metz RP Gustafson T Schedin P Porter WW 《Molecular and cellular biology》2008,28(6):1936-1946
30.
Fitzgerald J Xu X Pepper N Easterbrook M Solomon M 《Experimental & applied acarology》2008,44(4):293-306
Extensive sampling of strawberry plants in everbearing and June-bearing strawberry plantations and on potted plants showed
that different species of mites were spatially separated. Of the two phytophagous species recorded, Tetranychus urticae was most abundant on old leaves and Phytonemus pallidus on folded leaves and flower/fruit clusters. Predatory phytoseiid mites were found on all plant parts but different species
were spatially separated; Neoseiulus cucumeris and N. aurescens were found mostly on folded leaves and clusters, and N. californicus and Phytoseiulus persimilis on old and medium aged leaves. No Typhlodromus pyri were found in the field plantations. These patterns of distribution did not change over sampling dates in summer and early
autumn. An understanding of this within-plant zonation of mite species is important when studying predator–prey interactions
and when designing sampling strategies for strawberry. A programme to sample the entire mite system on strawberry should be
stratified to include all the above mentioned parts of the plant. Different sampling protocols, as appropriate, are required
for sampling different pest species and their associated predators. 相似文献