scholarly journals In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry

2020 ◽  
Vol 21 (13) ◽  
pp. 4649
Author(s):  
Sarah A. Overall ◽  
Lauren E. Price ◽  
Brice J. Albert ◽  
Chukun Gao ◽  
Nicholas Alaniva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dynamic Nuclear Polarization ◽  
Nuclear Polarization ◽  
Fluorescent Protein ◽  
Human Cell Line ◽  
Cellular Heterogeneity ◽  
Nmr Studies ◽  
Cellular Processes ◽  
Green Fluorescent ◽  
In Situ Detection

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.

scholarly journals Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms

2013 ◽  
Vol 11 (4) ◽  
pp. 629-635 ◽  
Author(s):  
L. M. Schaefer ◽  
V. S. Brözel ◽  
S. N. Venter
Keyword(s):  
Drinking Water ◽  
Green Fluorescent Protein ◽  
Health Risk ◽  
Salmonella Typhimurium ◽  
Fluorescent Protein ◽  
Laboratory Scale ◽  
Green Fluorescent ◽  
In Situ Detection

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.

scholarly journals Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Takuya Tomita ◽  
Shoshiro Hirayama ◽  
Yasuyuki Sakurai ◽  
Yuki Ohte ◽  
Hidehito Yoshihara ◽  
...  
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Embryonic Fibroblasts ◽  
Α Subunit ◽  
Biochemical Alterations ◽  
Cellular Processes ◽  
Responsible Gene ◽  
Intracellular Protein Degradation ◽  
Green Fluorescent

ABSTRACT The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α′ subunit of casein kinase II (CK2α′) as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

scholarly journals Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase IIα Mutant Human Cell Line

2004 ◽  
Vol 15 (12) ◽  
pp. 5700-5711 ◽  
Author(s):  
Adam J. Carpenter ◽  
Andrew C.G. Porter
Keyword(s):  
Cell Line ◽  
Human Cell ◽  
Fluorescent Protein ◽  
Human Cell Line ◽  
Chromosome Condensation ◽  
Mitotic Chromosomes ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Green Fluorescent ◽  
Dna Topoisomerase Iiα

DNA Topoisomerase IIα (topoIIα) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIα expression can be silenced in all cells by the addition of tetracycline. TopoIIα-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIα fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIα as an indicator for endogenous topoIIα dynamics, our analyses provide new evidence that topoIIα plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIβ and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

scholarly journals Dual Reporter System for In Situ Detection of Plasmid Transfer under Aerobic and Anaerobic Conditions

2010 ◽  
Vol 76 (13) ◽  
pp. 4553-4556 ◽  
Author(s):  
Jaroslaw E. Król ◽  
Linda M. Rogers ◽  
Stephen M. Krone ◽  
Eva M. Top
Keyword(s):  
Fluorescent Protein ◽  
Plasmid Transfer ◽  
Reporter System ◽  
T7 Promoter ◽  
Dual Reporter ◽  
Green Fluorescent ◽  
In Situ Detection ◽  
Aerobic And Anaerobic ◽  
Rna Polymerase Gene

ABSTRACT We designed a new genetic tool to detect plasmid transfer under anaerobic and aerobic conditions. The system is based on the T7 RNA polymerase gene and a T7 promoter-driven oxygen-independent green fluorescent protein, evoglow, alone or in combination with red fluorescent protein DsRed. Constructs are available as plasmids and mini-mariner transposons.

scholarly journals Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

1999 ◽  
Vol 67 (12) ◽  
pp. 6695-6697 ◽  
Author(s):  
Stephan Köhler ◽  
Safia Ouahrani-Bettache ◽  
Marion Layssac ◽  
Jacques Teyssier ◽  
Jean-Pierre Liautard
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Gene Fusion ◽  
Fluorescent Protein ◽  
Fusion System ◽  
Chromosomal Dna ◽  
Brucella Suis ◽  
Green Fluorescent ◽  
Inducible Genes

ABSTRACT A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA togfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

scholarly journals High-Titer Retroviral Vectors Containing the Enhanced Green Fluorescent Protein Gene for Efficient Expression in Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3316-3321 ◽  
Author(s):  
Ana Limón ◽  
Javier Briones ◽  
Teresa Puig ◽  
Mercé Carmona ◽  
Oscar Fornas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Green Fluorescent Protein Gene ◽  
Efficient System ◽  
Green Fluorescent

Abstract Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34+ cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

scholarly journals Dynamic Nuclear Polarization of Biomembrane Assemblies

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1246
Author(s):  
Nhi T. Tran ◽  
Frédéric Mentink-Vigier ◽  
Joanna R. Long
Keyword(s):  
Membrane Proteins ◽  
Dynamic Nuclear Polarization ◽  
Nuclear Polarization ◽  
Atomic Scale ◽  
Nuclear Spins ◽  
Nmr Studies ◽  
Nmr Characterization ◽  
Recent Developments ◽  
Lipid Assemblies

While atomic scale structural and dynamic information are hallmarks of nuclear magnetic resonance (NMR) methodologies, sensitivity is a fundamental limitation in NMR studies. Fully exploiting NMR capabilities to study membrane proteins is further hampered by their dilution within biological membranes. Recent developments in dynamic nuclear polarization (DNP), which can transfer the relatively high polarization of unpaired electrons to nuclear spins, show promise for overcoming the sensitivity bottleneck and enabling NMR characterization of membrane proteins under native-like conditions. Here we discuss fundamental aspects of DNP-enhanced solid-state NMR spectroscopy, experimental details relevant to the study of lipid assemblies and incorporated proteins, and sensitivity gains which can be realized in biomembrane-based samples. We also present unique insights which can be gained from DNP measurements and prospects for further development of the technique for elucidating structures and orientations of membrane proteins in native lipid environments.

Fluorescent reporters for the ubiquitin–proteasome system

2005 ◽  
Vol 41 ◽  
pp. 113-128 ◽  
Author(s):  
Florian A. Salomons ◽  
Lisette G.G.C. Verhoef ◽  
Nico P. Dantuma
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ubiquitin Proteasome System ◽  
Fluorescent Reporters ◽  
Cellular Processes ◽  
The Status ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent ◽  
Specific Degradation

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin–proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.

scholarly journals In Vitro and In Vivo Characterization of a Dual-Function Green Fluorescent Protein–HSV1-Thymidine Kinase Reporter Gene Driven by the Human Elongation Factor 1α Promoter

2002 ◽  
Vol 1 (2) ◽  
pp. 153535002002011
Author(s):  
Gary D. Luker ◽  
Kathryn E. Luker ◽  
Vijay Sharma ◽  
Christina M. Pica ◽  
Julie L. Dahlheimer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Elongation Factor ◽  
Dual Function ◽  
Elongation Factor 1Α ◽  
Green Fluorescent ◽  
Human Elongation Factor

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1α (EF-1α-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) ≈ 8-[3H]penciclovir (8-[3H]PCV) < 2′-fluoro-2′-deoxy-5-iodouracil-beta-d-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

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