The Fate of Host and Graft Cells in Early Healing of Bone Tunnel after Tendon Graft

2005 ◽  
Vol 33 (12) ◽  
pp. 1892-1897 ◽  
Author(s):  
Masashi Kobayashi ◽  
Nobuyoshi Watanabe ◽  
Yasushi Oshima ◽  
Yoshiteru Kajikawa ◽  
Mitsuhiro Kawata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Tendon Graft ◽  
Bone Tunnel ◽  
Host Cells ◽  
Wild Type ◽  
Group A ◽  
Group B ◽  
Green Fluorescent ◽  
Autologous Tendon Graft

Background The behavior of host and graft cells during the healing process after autologous tendon graft has not been elucidated. Hypothesis Host cells will integrate into the bone-tendon interface and contribute to cellular repopulation of the graft. Study Design Controlled laboratory study. Methods Twelve-week-old, genetically identical, female green fluorescent protein transgenic rats (n = 20) and wild-type rats (n = 20) were used. The rats were divided into 2 experimental groups. In group A, the Achilles tendons of wild-type rats were harvested and transplanted into the transcondylar femoral bone tunnels of green fluorescent protein rats. In group B, the Achilles tendons of green fluorescent protein rats were transplanted into a transcondylar femoral bone tunnel of wild-type rats. Immediately after transplantation (time zero) and at 1, 2, and 4 weeks after the transplantation, distal femoral epiphyses were harvested and cut into 14-μm serial sagittal frozen sections. The sections were examined with a confocal laser-scanning microscope to quantify green fluorescent protein-positive cell survival. Results At time zero, only host cells in group A and only graft cells in group B demonstrated green fluorescent protein signals. At 1 week in group A, many green fluorescent protein-positive cells were found in the graft. In group B, a few green fluorescent protein-positive cells were found in the graft. At 2 and 4 weeks in group A, many green fluorescent protein-positive cells were detected in the graft, but green fluorescent protein-positive cells had disappeared completely in group B. Conclusion Host cells, rather than graft cells, contribute to repair of the bone-tendon interface and the remodeling of grafts after simulated autologous tendon graft.

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.

Limitations on the use of fused green fluorescent protein to investigate structure–function relationships for the cauliflower mosaic virus movement protein

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1851-1855 ◽  
Author(s):  
Carole L. Thomas ◽  
Andrew J. Maule
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Cauliflower Mosaic Virus ◽  
Virus Movement ◽  
Wild Type ◽  
Tubule Formation ◽  
Mouth Disease Virus ◽  
Green Fluorescent

To investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP–MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.

scholarly journals A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants

2003 ◽  
Vol 373 (2) ◽  
pp. 403-408 ◽  
Author(s):  
Nadya G. GURSKAYA ◽  
Arkady F. FRADKOV ◽  
Natalia I. POUNKOVA ◽  
Dmitry B. STAROVEROV ◽  
Maria E. BULINA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Green Luminescence ◽  
Mammalian Cell Lines ◽  
Biological Tool ◽  
Fluorescent Pattern ◽  
Green Fluorescent

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu222→Gly (E222G) substitution, which removed the evolutionarily invariant Glu222. Re-introduction of Glu222 into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of ‘humanized’ aceGFP and β-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

scholarly journals Analysis of Localization of Mutated Tissue-Nonspecific Alkaline Phosphatase Proteins Associated with Neonatal Hypophosphatasia Using Green Fluorescent Protein Chimeras1

1998 ◽  
Vol 83 (11) ◽  
pp. 3936-3942
Author(s):  
Guiming Cai ◽  
Toshimi Michigami ◽  
Takehisa Yamamoto ◽  
Natsuo Yasui ◽  
Kenichi Satomura ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enzymatic Activity ◽  
Fluorescent Protein ◽  
Clinical Severity ◽  
Wild Type ◽  
Neonatal Patient ◽  
Tissue Nonspecific Alkaline Phosphatase ◽  
Green Fluorescent

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458–4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

scholarly journals SLAM (CD150)-Independent Measles Virus Entry as Revealed by Recombinant Virus Expressing Green Fluorescent Protein

2002 ◽  
Vol 76 (13) ◽  
pp. 6743-6749 ◽  
Author(s):  
Koji Hashimoto ◽  
Nobuyuki Ono ◽  
Hironobu Tatsuo ◽  
Hiroko Minagawa ◽  
Makoto Takeda ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Infected Individual ◽  
Lymphocyte Activation ◽  
Wild Type ◽  
Low Efficiency ◽  
Histopathological Studies ◽  
Green Fluorescent

ABSTRACT Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.

The effect of pressure on the excited-state proton transfer in the wild-type green fluorescent protein

2008 ◽  
Vol 455 (4-6) ◽  
pp. 303-306 ◽  
Author(s):  
Pavel Leiderman ◽  
Dan Huppert ◽  
S. James Remington ◽  
Laren M. Tolbert ◽  
Kyril M. Solntsev
Keyword(s):  
Excited State ◽  
Green Fluorescent Protein ◽  
Proton Transfer ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Effect Of Pressure ◽  
Excited State Proton Transfer ◽  
In The Wild ◽  
Green Fluorescent

Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium

2000 ◽  
Vol 113 (12) ◽  
pp. 2253-2265 ◽  
Author(s):  
M. Dumontier ◽  
P. Hocht ◽  
U. Mintert ◽  
J. Faix
Keyword(s):  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Fluorescent Protein ◽  
Colony Growth ◽  
Dominant Negative ◽  
Related Protein ◽  
Wild Type ◽  
Equal Capacity ◽  
Green Fluorescent

The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.

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