High-Sensitivity Real-Time Analysis of Nanoparticle Toxicity in Green Fluorescent Protein-Expressing Zebrafish

Small ◽  
2012 ◽  
Vol 9 (6) ◽  
pp. 863-869 ◽  
Author(s):  
Yu Pan ◽  
Annika Leifert ◽  
Michael Graf ◽  
Frank Schiefer ◽  
Sven Thoröe-Boveleth ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Time Analysis ◽  
Nanoparticle Toxicity ◽  
Real Time Analysis ◽  
Green Fluorescent

Real-time analysis of GLUT4 trafficking in single living cells using green-fluorescent protein

1997 ◽  
Vol 25 (3) ◽  
pp. 460S-460S
Author(s):  
J.M. Tavaré ◽  
S.P. Dobson ◽  
C. Livingstone ◽  
A.A. Culbert ◽  
P.B. Oatey ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Green Fluorescent ◽  
Single Living Cells ◽  
Single Living

scholarly journals Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

2006 ◽  
Vol 50 (8) ◽  
pp. 2806-2813 ◽  
Author(s):  
T. Ueno ◽  
Y. Eizuru ◽  
H. Katano ◽  
T. Kurata ◽  
T. Sata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Fluorescent Protein ◽  
Clinical Isolates ◽  
Pml Bodies ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

scholarly journals Real-Time Optical Monitoring of Ligand-Mediated Internalization of α1b-Adrenoceptor with Green Fluorescent Protein

1998 ◽  
Vol 12 (8) ◽  
pp. 1099-1111 ◽  
Author(s):  
Takeo Awaji ◽  
Akira Hirasawa ◽  
Masakazu Kataoka ◽  
Hitomi Shinoura ◽  
Yasuhisa Nakayama ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Optical Monitoring ◽  
Green Fluorescent

scholarly journals Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

2000 ◽  
Vol 97 (13) ◽  
pp. 7237-7242 ◽  
Author(s):  
M. F. Garcia-Parajo ◽  
G. M. J. Segers-Nolten ◽  
J.-A. Veerman ◽  
J. Greve ◽  
N. F. van Hulst
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Protein Molecules ◽  
Green Fluorescent

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

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