A Green-emitting Fluorescent Protein from Galaxeidae Coral and Its Monomeric Version for Use in Fluorescent Labeling

Karasawa, Satoshi; Araki, Toshio; Yamamoto-Hino, Miki; Miyawaki, Atsushi

doi:10.1074/jbc.m304063200

Cited by 184 publications

(136 citation statements)

References 11 publications

(18 reference statements)

Supporting

Mentioning

132

Contrasting

Order By: Relevance

“…Concurrently, 10 6 per ml target cells were stained with PKH67 using a MINI67 cell linker kit (Sigma). 22 After the PKH67-stained target cells (PKH67 þ ) were incubated in the presence or absence of CMC-544 containing 5 ng/ml calichemicin DMH or equivalent amount of G5/44 for two hours and washed three times, they were co-cultured with 10 4 per ml natural killer cells in triplicate with or without 2 mg/ml rituximab for 4 h at 37 1C. Then the cells were stained with 0.1 mg/ml PI solution and analyzed by flow cytometry.…”

Section: Antibody-dependent Cellular Cytotoxicity Assaysmentioning

confidence: 99%

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells

et al. 2009

View full text Add to dashboard Cite

We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (Po0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.

show abstract

Section: Antibody-dependent Cellular Cytotoxicity Assaysmentioning

confidence: 99%

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells

et al. 2009

View full text Add to dashboard Cite

show abstract

“…To render this interface even more hydrophilic, we introduced the additional substitution V123T. A homologous mutation was also introduced recently in the monomerization of Azami green (20). Size-exclusion chromatography revealed a M W of Ϸ40 kDa for this variant at 10 M concentration of monomer subunits, which is between the M W values for tetrameric DsRed and monomeric GFP (Fig.…”

Section: Resultsmentioning

confidence: 99%

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

Wiedenmann

Ivanchenko

Oswald

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

634

674

View full text Add to dashboard Cite

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at Ϸ390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Fö rster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.

show abstract

“…While two fluorescent proteins used in those previous studies had different excitation wavelengths, AG and DsRed2 have the same excitation wavelength of 488 nm. AG emits brighter green fluorescence than jellyfish green fluorescent protein (GFP) and the brightest among commercial green fluorescent proteins (12). It requires a shorter period for maturation than GFP, allowing us to perform an assay procedure in a short period of time.…”

Section: Original Articlementioning

confidence: 99%

“…In this study we applied flow cytometry to the IRES studies, in an attempt to simplify the assay procedure and to expand the scope of the study. Thus, we used a combination of two fluorescent proteins, DsRed2 (11) and Azami-Green (AG) (12), which are excited by the light of the identical wave length and whose emission lights are easily discriminated. We constructed and used a bicistronic reporter plasmid with genes encoding these two fluorescent proteins, and by flow cytometry, quantitatively determined fluorescent signals in individual cells transfected with the plasmid.…”

mentioning

confidence: 99%

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

et al. 2011

View full text Add to dashboard Cite

The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5 0 -end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs. ' 2011 International Society for Advancement of Cytometry Key terms hepatitis C virus; internal ribosome entry site; DsRed2; Azami Green; flow cytometry HEPATITIS C virus (HCV) is a major causative agent of liver cirrhosis and hepatocellular carcinoma (1). The HCV genome is a plus-strand RNA consisting of $9,600 nucleotides (nt) and has a single long open reading frame encoding a polyprotein of about 3,000 amino acids (2). Unlike the translation of eukaryotic mRNA starting from the 5 0 -cap structure, the translation of HCV RNA initiates at the internal ribosome entry site (IRES), which encompasses most of the 5 0 -untranslated region (5 0 -UTR) and about 40 nt of the core protein-coding region (3,4). The HCV IRES is highly conserved among HCV isolates in both the primary sequence and secondary structure (5-7). Since the translation is an essential step in the viral replication and possibly in the pathogenic functions, it would be important to evaluate the activity of HCV IRES for such studies.The HCV IRES activity has been studied, by using biochemical methods with luciferase genes as reporters (8-10), which have some flaws resulting from using cell lysates. In this study we applied flow cytometry to the IRES studies, in an attempt to simplify the assay procedure and to expand the scope of the study. Thus, we used a combination of two fluorescent proteins, DsRed2 (11) and Azami-Green (AG) (12), which are excited by the light of the identical wave length and whose emission lights are easily discriminated. We constructed and used a bicistronic reporter plasmid with genes encoding these two fluorescent proteins, and by flow cytometry, quantitatively determined fluorescent signals in individual cells transfected with the plasmid. To

show abstract

A Green-emitting Fluorescent Protein from Galaxeidae Coral and Its Monomeric Version for Use in Fluorescent Labeling

Cited by 184 publications

References 11 publications

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

Contact Info

Product

Resources

About