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At least 199 records · Page 11Linked to original sources
PubMed2001

Direct measurement of conformational changes on DNA molecule intercalating with a fluorescence dye in an electrophoretic buffer solution by means of atomic force microscopy.

By means of atomic force microscopy (AFM), we performed the direct imaging of DNA molecules (200, 500, 1000 bp) in a Tris-borate buffer solution, and measured the contour length and the end-to-end distance of DNA. Processing the data according to the worm-like chain model, we calculated the persistence length of the double-stranded DNA. Based on the analysis of the contour length and the persistence length, we discussed the interactions between DNA and an intercalating fluorescence dye (YO-PRO-1). YO-PRO-1 stacks between the base pairs and extends the contour length of DNA, changing the electric charge and the persistence length of DNA. From AFM measurement, we investigated directly the relationship between the persistence length and the number of the YO-PRO-1 intercalating to DNA. We will discuss on the relationship between the effect of an intercalating dye on the electrophoretic behavior and the conformational changes of DNA with an intercalating dye.

Benzoxazoles↗
PubMed2003

Oxidation and degradation products of papaverine. Part II[1]: investigations on the photochemical degradation of papaverine solutions.

The structure of the final degradation product formed in papaverine solutions in either water or chloroform was found to be a 2, 3, 9, 10-tetramethoxy-12-oxo-12H-indolo [2, 1-a] isoquinolinylium salt (a dibenzo [b, g] pyrrocolonium derivative). Its formation from papaverine oxidation products that is papaverinol, papaveraldine, and papaverine-N-oxide chloroform solutions under the influence of UV light, was investigated and possible reaction pathways are discussed.

Chemistry, Pharmaceutical↗
PubMed2002

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay.

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.

Benzoxazoles↗
PubMed1995

Characterization of the binding of YO to [poly(dA-dT)]2 and [poly(dG-dC)]2, and of the fluorescent properties of YO and YOYO complexed with the polynucleotides and double-stranded DNA.

The interaction between the fluorescent dye YO (oxazole yellow) and the alternating polynucleotides [poly(dA-dT)]2 [the duplex of alternating poly(dA-dT)] and [poly(dG-dC)]2 [the duplex of alternating poly(dG-dC)] has been studied with optical spectroscopic techniques including absorbance, flow linear dichroism, CD, and fluorescence measurements. The principal features of the spectra are very similar for the two polynucleotide solutions, showing that YO binds quite similarly to AT and GC base pairs. From a strongly negative reduced linear dichroism (LDr) in the dye absorption band, an induced negative CD, and transfer of energy from the bases to bound YO, we conclude that at low mixing ratios YO is intercalated in both [poly(dA-dT)]2 and [poly(dG-dC)]2. At higher mixing ratios an external binding mode starts to contribute, evidenced from the appearance of an exciton CD. The conclusion that YO binds in a similar way to AT and GC base pairs should be valid also for the dimer YOYO since its YO units have been found to bind to double-stranded (dsDNA) in the same way as the YO monomer. The fluorescence properties of YO and YOYO complexed with DNA or the polynucleotides have been characterized by studying the dependence of fluorescence intensity on temperature, mixing ratio, and ionic strength. The fluorescence intensity and fluorescence lifetime of YO-DNA decrease strongly with increasing mixing ratio, whereas the fluorescence intensity of YOYO-DNA shows a weaker dependence, indicating that the quantum yield depends on the distance between the YO chromophores on the DNA chain. Further, the fluorescence intensity of YO depends on the base sequence; the quantum yield and fluorescence lifetime for YO complexed with [poly(dG-dC)]2 are about twice as large as for YO complexed with [poly(dA-dT)]2. Measurements of excitation spectra at different mixing ratios and different emission wavelengths indicate that the fluorescence of the externally bound chromophores is negligible compared to the intercalated ones.

Benzoxazoles↗
PubMed1994

TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry.

Flow cytometric (FCM) studies were performed on nuclei, ethanol-fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase-treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G2/M phases of the cell cycle. Ethanol-fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wave-lengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA.

Animals↗
PubMed1994

Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis.

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime.

DNA↗
PubMed1994

Highly sensitive, specific, and stable new fluorescent DNA stains for confocal laser microscopy and image processing of normal paraffin sections.

The area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three-dimensional (3-D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO-1 iodide and YOYO-1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO-1 iodide and YOYO-1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO-1 iodide and YOYO-1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO-1 iodide and YOYO-1 iodide. Contrary to acriflavine, bleaching of TOTO-1 iodide and YOYO-1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acriflavine↗
PubMed2003

Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: interest for cell death investigations.

BACKGROUND: Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Deltapsimt) and membrane integrity fluorochromes. Rhodamine 123 and DiOC6(3) remain controversial to identify cells displaying a low Deltapsimt. JC-1 constitutes a good Deltapsimt indicator, due to a fluorescence shift from green to orange emission, according to the increase in Deltapsimt. Nevertheless, it is not feasible to analyze it simultaneously with propidium iodide. Among available fluorescent probes, TOTO-3 seems to be a good candidate for double staining with JC-1. METHODS: Cell death of HaCaT cells was induced by H2O2 and FasL. Samples were stained with DiOC6(3)/IP or JC-1/TOTO-3 then analyzed by flow cytometry. Results were supported by confocal microscopy analyses of mitochondrial membrane potential. Moreover, cell morphology was determined on the sorted subpopulations defined on the basis of staining (JC-1 versus TOTO-3). RESULTS: We found that JC-1 is a more efficient mitochondrial probe than DiOC6(3). After stress induction, the fluorescence level of JC-1 and TOTO-3 clearly defined three fluorescent subpopulations, respectively: (1) JC-1high and TOTO-3low, (2) JC-1low and TOTO-3medium, and (3) JC-1low and TOTO-3high. Their morphologic aspects after cell sorting indicated that they corresponded to three functional states (intact, apoptotic, and necrotic cells), and data were supported by caspase activity measurements. CONCLUSIONS: We propose a reliable and efficient staining, with JC-1 and TOTO-3 to discriminate three functional cellular states: intact, apoptotic, and necrotic/late apoptotic cells by flow cytometry.

Apoptosis↗
PubMed2005

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.

BACKGROUND: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. METHODS: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. RESULTS: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap. CONCLUSIONS: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.

Cell Line, Tumor↗
PubMed2005

Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.

BACKGROUND: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. METHODS: Samples of blood from Plasmodium yoelii-infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. RESULTS: The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. CONCLUSIONS: The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria.

Animals↗
PubMed2006

Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis.

BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. METHODS: The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. RESULTS: Tissues as thick as 500 microm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). CONCLUSIONS: The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-microm thick.

Amines↗
PubMed1996

Influence of optical probing with YOYO on the electrophoretic behavior of the DNA molecule.

The influence of the fluorescent dye YOYO (1,1'-(4,4,8,8,-tetramethyl- 4,8-diazaundecamethylene)bis[4-[[3-methyl-benzo-1,3-oxazol-2 -yl] methylidene]-1,4-dihydroquinolinium] tetraiodide) on the electrophoretic behavior of the DNA molecule was investigated. This is important when using YOYO as a probe in capillary electrophoresis or in fluorescence microscopy studies of DNA with the purpose of studying the migration mechanism of DNA on the molecular level. We have measured the mobility and orientation dynamics (using the linear dichroism technique) for both pure DNA and the YOYO-DNA complex in agarose gel in order to compare their electrophoretic properties. Mobility decreases, the degree of orientation becomes lower, and the orientational dynamics slower, when YOYO binds to DNA. However, the dependence on field strength of the mobility, orientation and orientational dynamics, are similar for DNA and YOYO-DNA, indicating that the mode of migration does not change significantly upon binding YOYO to DNA. Furthermore, since our results show that the effect of YOYO on both the degree of orientation and orientational dynamics of the DNA can be measured and therefore be compensated for, it can be concluded that YOYO is an excellent optical probe for the study of the migrational behavior of DNA.

Bacteriophage T7↗
PubMed1996

Field and pore size dependence of the electrophoretic mobility of DNA: a combination of fluorescence recovery after photobleaching and electric birefringence measurements.

By combining an electrophoretic cell with a setup of fluorescence recovery after photobleaching (FRAP) we can measure the electrophoretic mobility mu of double-stranded lambda DNA in agarose gel as a function of electric field E and gel concentration C. Mobility varies linearly with the field in agreement with the biased reptation model with fluctuations. The slopes are analyzed in term of orientation and compared with birefringence results. The mobility extrapolated at zero field follows the prediction of the reptation theory; we deduced the variation of the pore size with the agarose concentration. With a special use of our setup, we measure directly the free-mobility mu 0 of the DNA.

Bacteriophage lambda↗
PubMed2003

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection.

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.

Bacteriophage phi X 174↗
PubMed2005

The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes.

Extracellular nucleotide di- and triphosphates such as ATP and ADP mediate their effects through purinergic P2 receptors belonging to either the metabotropic P2Y or the ionotropic P2X receptor family. The P2X7R is a unique member of the P2X family, which forms a pore in response to ligand stimulation, regulating cell permeability, cytokine release, and/or apoptosis. This receptor is also unique in that its affinity for the ligand benzoyl-benzoyl ATP (BzATP) is at least 10-fold greater than that of ATP. Primary human fetal astrocytes in culture express low-levels of P2X7R mRNA and protein, and BzATP induces only a slight influx in intracellular calcium [Ca2+]i, with little demonstrable effect on gene expression or pore formation in these cells. We now show that, following treatment with the proinflammatory cytokine IL-1beta, BzATP induces a robust rise in [Ca2+]i with agonist and antagonist profiles indicative of the P2X7R. IL-1beta also induced the formation of membrane pores as evidenced by the uptake of YO-PRO-1 (375 Da). Quantitative real-time PCR demonstrated transient upregulation of P2X7R mRNA in IL-1beta-treated cells, while FACS analysis indicated a similar upregulation of P2X7R protein at the cell membrane. In multiple sclerosis lesions, immunoreactivity for the P2X7R was demonstrated on reactive astrocytes in autopsy brain tissues. In turn, P2X7R stimulation increased the production of IL-1-induced nitric oxide synthase activity by astrocytes in culture. These studies suggest that signaling via the P2X7R may modulate the astrocytic response to inflammation in the human central nervous system.

Adenosine Triphosphate↗