scholarly journals An Influence of Modification with Phosphoryl Guanidine Combined with a 2′-O-Methyl or 2′-Fluoro Group on the Small-Interfering-RNA Effect

2021 ◽  
Vol 22 (18) ◽  
pp. 9784
Author(s):  
Anna S. Pavlova ◽  
Kristina I. Yakovleva ◽  
Anna V. Epanchitseva ◽  
Maxim S. Kupryushkin ◽  
Inna A. Pyshnaya ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Biochemical Properties ◽  
Rnase A ◽  
Snake Venom Phosphodiesterase ◽  
Passenger Strand ◽  
Green Fluorescent ◽  
Uv Melting ◽  
Interfering Rna

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2′-O-methyl (2′-OMe) or PG/2′-fluoro (2′-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

scholarly journals Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa andSalmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 861-870 ◽  
Author(s):  
A. Alev Gerçeker ◽  
Tanweer Zaidi ◽  
Peter Marks ◽  
David E. Golan ◽  
Gerald B. Pier
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bacterial Uptake ◽  
Cftr Protein ◽  
Green Fluorescent

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry ofPseudomonas aeruginosa and Salmonella entericaserovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

1999 ◽  
Vol 112 (16) ◽  
pp. 2705-2714
Author(s):  
E.M. Burns ◽  
L. Christopoulou ◽  
P. Corish ◽  
C. Tyler-Smith
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Quantitative Measurement ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Chromosome Loss ◽  
Facs Analysis ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

2003 ◽  
Vol 284 (5) ◽  
pp. H1647-H1654 ◽  
Author(s):  
Jean-Philippe Fortin ◽  
Johanne Bouthillier ◽  
François Marceau
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Brefeldin A ◽  
Metabolic Inhibitors ◽  
Maximal Effect ◽  
Hek 293 ◽  
B1 Receptors ◽  
Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

scholarly journals Functional Analysis of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Differentially Expressed by Variants of Human HT-1080 Fibrosarcoma Exhibiting High and Low Levels of Intravasation and Metastasis

2007 ◽  
Vol 282 (49) ◽  
pp. 35964-35977 ◽  
Author(s):  
Juneth J. Partridge ◽  
Mark A. Madsen ◽  
Veronica C. Ardi ◽  
Thales Papagiannakopoulos ◽  
Tatyana A. Kupriyanova ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Cancer Cell ◽  
Small Interfering Rna ◽  
Cultured Cells ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Down Regulation ◽  
Primary Tumors ◽  
Tissue Inhibitors ◽  
Interfering Rna

The role of tumor-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in cancer cell dissemination was analyzed by employing two variants of human HT-1080 fibrosarcoma, HT-hi/diss and HT-lo/diss, which differ by 50-100-fold in their ability to intravasate and metastasize in the chick embryo. HT-hi/diss and HT-lo/diss were compared by quantitative reverse transcription-PCR and Western blot analyses for mRNA and protein expression of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3) in cultured cells in vitro and in primary tumors in vivo. MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, both in cultures and in tumors, whereas the HT-lo/diss variant consistently expressed higher levels of MMP-2, TIMP-1, and TIMP-2. Small interfering RNA-mediated down-regulation of MMP-2 and TIMP-2 increased intravasation of HT-lo/diss cells. Coordinately, treatment of the developing HT-hi/diss tumors with recombinant TIMP-1 and TIMP-2 significantly reduced HT-hi/diss cell intravasation. However, a substantial increase of HT-hi/diss dissemination was observed upon small interfering RNA-mediated down-regulation of three secreted MMPs, including the interstitial collagenase MMP-1 and the two gelatinases, MMP-2 and MMP-9, but not the membrane-tethered MMP-14. The addition of recombinant pro-MMP-9 protein to the HT-hi/diss tumors reversed the increased intravasation of HT-hi/diss cells, in which MMP-9 was stably down-regulated by short hairpin RNA interference. This rescue did not occur if the pro-MMP-9 was stoichiometrically complexed with TIMP-1, pointing to a direct role of the MMP-9 enzyme in regulation of HT-hi/diss intravasation. Collectively, these findings demonstrate that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. not promoting but rather catalytically interfering with the early stages of cancer dissemination.

scholarly journals Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing

2019 ◽  
Vol 5 (8) ◽  
pp. eaax0801 ◽  
Author(s):  
Minh Khanh Nguyen ◽  
Cong Truc Huynh ◽  
Alex Gilewski ◽  
Samantha E. Wilner ◽  
Keith E. Maier ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Sirna Delivery ◽  
Hydrolytic Degradation ◽  
Nucleic Acid Delivery ◽  
Covalent Bonds ◽  
Disulfide Linkages ◽  
Green Fluorescent ◽  
Interfering Rna ◽  
Transfection Agent

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein–expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

scholarly journals Characterization of a Neuraminidase-Deficient Influenza A Virus as a Potential Gene Delivery Vector and a Live Vaccine

2004 ◽  
Vol 78 (6) ◽  
pp. 3083-3088 ◽  
Author(s):  
Kyoko Shinya ◽  
Yutaka Fujii ◽  
Hiroshi Ito ◽  
Toshihiro Ito ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Mutant Cell ◽  
Wild Type Virus ◽  
Reading Frame ◽  
Foreign Genes ◽  
Green Fluorescent

ABSTRACT We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >106 PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with ≥105 PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.

scholarly journals Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

2002 ◽  
Vol 156 (3) ◽  
pp. 511-518 ◽  
Author(s):  
Pierre Barbero ◽  
Lenka Bittova ◽  
Suzanne R. Pfeffer
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Late Endosome ◽  
Live Cells ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network ◽  
Protein Variants

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

scholarly journals Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3)

2005 ◽  
Vol 389 (2) ◽  
pp. 355-364 ◽  
Author(s):  
Markus Napirei ◽  
Swantje Wulf ◽  
Dirk Eulitz ◽  
Hans Georg Mannherz ◽  
Thomas Kloeckl
Keyword(s):  
Fusion Proteins ◽  
Secretory Pathway ◽  
Nuclear Dna ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Dnase Γ ◽  
3T3 Fibroblasts ◽  
Green Fluorescent

Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase γ, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed ‘naked’ plasmid DNA, whereas Dnase1l3 cleaved nuclear DNA with high activity. Dnase1l3 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase1l3-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.

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