scholarly journals Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Yi-Shin Pan ◽  
Hung-Ju Wei ◽  
Chung-Chieh Chang ◽  
Chung-Hung Lin ◽  
Ting-Shyang Wei ◽  
...  
Keyword(s):  
Insect Cell ◽  
Matrix Protein ◽  
Green Fluorescence Protein ◽  
Structural Rearrangement ◽  
Jurkat Cells ◽  
Proton Channel ◽  
Receptor Interaction ◽  
Transmission Electron ◽  
Fluorescence Protein

We have constructed virus-like particles (VLPs) harboring hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) ,and proton channel protein (M2) using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP) was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

scholarly journals A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection

2018 ◽  
Vol 23 (8) ◽  
pp. 869-876
Author(s):  
Bendix R. Slegtenhorst ◽  
Oscar R. Fajardo Ramirez ◽  
Yuzhi Zhang ◽  
Zahra Dhanerawala ◽  
Stefan G. Tullius ◽  
...  
Keyword(s):  
Vascular Endothelium ◽  
Critical Role ◽  
Green Fluorescence Protein ◽  
Dependent Manner ◽  
Reporter System ◽  
Health And Disease ◽  
Fluorescence Protein ◽  
Biomechanical Stimuli ◽  
Dose Dependent

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the “pharmacomimetics of atheroprotective flow” on the vascular endothelium.

scholarly journals Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

2007 ◽  
Vol 73 (17) ◽  
pp. 5411-5420 ◽  
Author(s):  
Yu-Sin Jang ◽  
Young Ryul Jung ◽  
Sang Yup Lee ◽  
Ji Mahn Kim ◽  
Jeong Wook Lee ◽  
...  
Keyword(s):  
Succinic Acid ◽  
Shuttle Vector ◽  
Green Fluorescence Protein ◽  
Expression Vectors ◽  
Rumen Bacteria ◽  
Gene Encoding ◽  
Shuttle Vectors ◽  
Genes Encoding ◽  
Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells

2002 ◽  
Vol 282 (5) ◽  
pp. C1053-C1063 ◽  
Author(s):  
Jun Chen ◽  
Filip Braet ◽  
Sergey Brodsky ◽  
Talia Weinstein ◽  
Victor Romanov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Electrical Resistance ◽  
Polyclonal Antibodies ◽  
Endothelial Permeability ◽  
Green Fluorescence Protein ◽  
Vascular Endothelial ◽  
Transmission Electron ◽  
Glomerular Epithelial Cells ◽  
Fluorescence Protein ◽  
Endothelial Growth Factor

Glomerular epithelial cells (GEC) are a known site of vascular endothelial growth factor (VEGF) production. We established immortalized rat GEC, which retained the ability to produce VEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells cultured on GEC-conditioned matrix, an indicator of the permeability of monolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased by VEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showed that VEGF results in a rapid appearance of transcellular elongated structures decorated with caveolin. Transmission electron microscopy of endothelial cells showed that caveolae undergo rapid internalization and fusion 30 min after application of VEGF-165. Later (36 h), endothelial cells pretreated with VEGF developed fenestrae and showed a decrease in electrical resistance. Immunoelectron microscopy of glomeruli confirmed VEGF localization to podocytes and in the basement membrane. In summary, immortalized GEC retain the ability to synthesize VEGF. Matrix-deposited and soluble VEGF leads to the enhancement of caveolae expression, their fission and fusion, formation of elongated caveolin-decorated structures, and eventual formation of fenestrae, both responsible for the increase in endothelial permeability.

scholarly journals Purification and Characterization of 6xHis Tagged Green Fluorescence Protein (GFP)

2017 ◽  
Vol 4 (3) ◽  
pp. 027-032
Author(s):  
Nasir Anka Garba ◽  
◽  
Kabiru Usman ◽  
Dayo Oke Okusaga ◽  
Shehu Abdullahi ◽  
...  
Keyword(s):  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Purification And Characterization ◽  
Fluorescence Protein

scholarly journals Characterization of melanoidin formed from glucose with alanine  by Fluorescence spectroscopy and redox chemistry in vitro cells: تشخيص الميلانويدين المتشكل من الكلوكوز مع الألنين بمطياف الفلوره ودراسة تأثيره التأكسدي على خلايا CHO-K1 المختبرية

2020 ◽  
Vol 4 (1) ◽  
Author(s):  
Ghassan Faisal Mohsin, Abdulridha Ati Jaafar
Keyword(s):  
Fluorescence Spectroscopy ◽  
Chinese Hamster ◽  
Emission Spectra ◽  
Green Fluorescence Protein ◽  
Time Resolved ◽  
Yellow Fluorescence Protein ◽  
Fluorescence Protein ◽  
Dialysis Time

Possible identify and describe a polymer formed from Glc with Ala by Fluorescence spectroscopy. Melanoidins before dialysis was higher than those of melanoidin after dialysis. Time‐resolved fluorescence has been applied in the characterization of melanoidin and was observed. Melanoidins show a decrease in the absorption after the addition of hydrogen peroxide when the concentrations of H2O2 from 100 - 200 μl are used. However, the data suggests that the melanoidin did not penetrate into the cells (CHO-K1) after 1 and 2 hours, but it can be observed after 24 hours. Abbreviation: TRES, time-resolved emission spectra ; Glc, Glucose ; Ala, Alanine ; YFP, Yellow fluorescence protein ; GFP, green fluorescence protein ; CHO, Chinese hamster ovary

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