Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection.
AuthorsCornelius M, Wörth CG, Kliem HC, Wiessler M, Schmeiser HH
JournalElectrophoresis
PubMed ID15934055
'We investigated the separation and detection of the 5''-monophosphates of 2''-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5''-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5''-monophosphates (2''-deoxyguanosine-5''-monophosphate, 2''-deoxyadenosine-5''-monophosphate, 2''-deoxycytidine-5''-monophosphate, and thymidine-5''-monophosphate) were prepared and subjected to ... More
Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.
AuthorsLyko F, Stach D, Brenner A, Stilgenbauer S, Döhner H, Wirtz M, Wiessler M, Schmitz OJ
JournalElectrophoresis
PubMed ID15188237
'Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis ... More
Detection of lipid peroxidation on erythrocytes using the excimer-forming property of a lipophilic BODIPY fluorescent dye.
AuthorsMakrigiorgos GM
JournalJ Biochem Biophys Methods
PubMed ID9310865
'Lipophilic analogues of dipyrrometheneboron (BODIPY-FL) dyes, used for membrane studies, normally fluoresce in the green wavelength region (approximately 516 nm), but at high local concentration, they shift their emission to the red region (approximately 540-600 nm) via excimer formation. A two-wavelength-based method is described that utilizes the excimer-forming property of ... More
Antibody affinity maturation using bacterial surface display.
AuthorsDaugherty PS, Chen G, Olsen MJ, Iverson BL, Georgiou G
JournalProtein Eng
PubMed ID9796833
'A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 ... More
RNA analysis by MEKC with LIF detection.
AuthorsCornelius MG, Schmeiser HH,
JournalElectrophoresis
PubMed ID17922502
'We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5''-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5''-phosphate group using CE with LIF. BODIPY conjugates of ... More
Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection.
AuthorsWirtz M, Stach D, Kliem HC, Wiessler M, Schmitz OJ
JournalElectrophoresis
PubMed ID15004844
'An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2''-desoxyribonucleoside-3''-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out ... More
Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands.
AuthorsBaker JG, Middleton R, Adams L, May LT, Briddon SJ, Kellam B, Hill SJ,
JournalBr J Pharmacol
PubMed ID20105183
BACKGROUND AND PURPOSE: The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties ... More
Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation.
AuthorsFranco R, Panayiotidis MI, Cidlowski JA,
JournalJ Biol Chem
PubMed ID17724027
Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via ... More
Capillary electrophoretic analysis of genomic DNA methylation levels.
AuthorsStach D, Schmitz OJ, Stilgenbauer S, Benner A, Döhner H, Wiessler M, Lyko F
JournalNucleic Acids Res
PubMed ID12527791
Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene- specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established ... More
Capillary electrophoresis-laser induced fluorescence analysis of endogenous damage in mitochondrial and genomic DNA.
AuthorsWirtz M, Schumann CA, Schellenträger M, Gäb S, Vom Brocke J, Podeschwa MA, Altenbach HJ, Oscier D, Schmitz OJ
JournalElectrophoresis
PubMed ID15929058
Reactive oxygen molecules are formed in vivo as by-products of normal aerobic metabolism. All organisms dependent on oxygen are inevitably exposed to these species so that DNA damage can occur in both genomic and mitochondrial DNA (mtDNA). In order to determine endogenous DNA damage we have developed an analytical method ... More
Characterization of DNA demethylation effects induced by 5-Aza-2'-deoxycytidine in patients with myelodysplastic syndrome.
AuthorsMund C, Hackanson B, Stresemann C, Lübbert M, Lyko F
JournalCancer Res
PubMed ID16103056
Azanucleoside drugs such as 5-azacytidine (Vidaza) and 5-aza-2'-deoxycytidine (decitabine, Dacogen) function as DNA methyltransferase inhibitors in vitro and represent promising new drugs for the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia. In this study, we aimed to determine the effect of decitabine on the genomic methylation level in ... More
Chemical synthesis and biological activity of a novel fluorescent etoposide derivative.
AuthorsLazo JS, Li T, Woo ES, Settineri CE, Allan WP, Yalowich JC
JournalBiochem Pharmacol
PubMed ID9113091
The antineoplastic activity of etoposide resides in its ability to poison the nuclear enzyme DNA topoisomerase II (topo II). The factors that control the cellular entry and subcellular distribution of etoposide remain poorly understood. Therefore, we have synthesized a novel fluorescence-labeled etoposide (Bodipyetoposide) by coupling 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionylethylenediamine (Bodipy) to 4'-benzyloxycarbonyl-4'-demethylepipodophyllotoxin ... More
Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.
AuthorsDaugherty PS, Olsen MJ, Iverson BL, Georgiou G
JournalProtein Eng
PubMed ID10436088
Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia ... More
Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies.
AuthorsDaugherty PS, Chen G, Iverson BL, Georgiou G
JournalProc Natl Acad Sci U S A
PubMed ID10688877
Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) ... More