Cell-to-cell movement of potato spindle tuber viroid.
AuthorsDing B, Kwon MO, Hammond R, Owens R
JournalPlant J
PubMed ID9375403
Viroids are non-translatable, autonomously replicating circular RNAs that infect only plants. An important component of the viroid infection process is cell-to-cell movement; however, there is virtually no information available about the pathways and mechanisms of this process. In this study, potato spindle tuber viroid (PSTVd) has been used as a ... More
Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest.
AuthorsBennin David A; Don Aruni S Arachchige; Brake Tiffany; McKenzie Jennifer L; Rosenbaum Heidi; Ortiz Linette; DePaoli-Roach Anna A; Horne Mary C;
JournalJ Biol Chem
PubMed ID11956189
Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., ... More
Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis.
AuthorsPerandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici MC, Arcangeletti MC, Snounou G, Dettori G, Chezzi C
JournalJ Clin Microbiol
PubMed ID15004078
A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was ... More
Visualization of replication initiation and elongation in Drosophila.
AuthorsClaycomb JM, MacAlpine DM, Evans JG, Bell SP, Orr-Weaver TL
JournalJ Cell Biol
PubMed ID12403810
'Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of ... More
Solution structure and energy calculation of bis-intercalation of homodimeric thiazole orange dye derivatives in DNA: effects of modifying the linker.
AuthorsBondensgaard K, Jacobsen JP
JournalBioconjug Chem
PubMed ID10502338
'We have used two-dimensional (1)H NMR spectroscopy obtained at 750 MHz to determine a high-resolution solution structure of the double-stranded DNA oligonucleotide d(5''-CGCTAGCG-3'')(2) complexed with the bis-intercalating dye 1,1''-(5,5,9,9-tetramethyl-5, 9-diazatridecamethylene)-bis-4-[3-ethyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quino-linium tetraiodide (TOTO11Et). The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross-peaks ... More
Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression.
AuthorsKu NO, Michie S, Resurreccion EZ, Broome RL, Omary MB
JournalProc Natl Acad Sci U S A
PubMed ID11917136
'Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33-->Ala (S33A) K18. In S33A but not wild-type ... More
Isolation and characterization of a human STAT1 gene regulatory element. Inducibility by interferon (IFN) types I and II and role of IFN regulatory factor-1.
AuthorsWong LH, Sim H, Chatterjee-Kishore M, Hatzinisiriou I, Devenish RJ, Stark G, Ralph SJ
JournalJ Biol Chem
PubMed ID11909852
'The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs). STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors. Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have ... More
Heterodimeric DNA-binding dyes designed for energy transfer: stability and applications of the DNA complexes.
AuthorsBenson SC, Mathies RA, Glazer AN
JournalNucleic Acids Res
PubMed ID8284220
'Spectroscopic studies of the complexes of double-stranded (ds) DNA with the polymethylene-amine linked heterodimers thiazole orange-thiazole blue, thiazole orange-ethidium, and fluorescein-ethidium, in each case show efficient energy transfer from donor to acceptor chromophores (Benson, S.C., Singh, P. and Glazer, A.N. (1993) accompanying manuscript). A quantitative assay of the stability of ... More
Differential effects of deuterium oxide on the fluorescence lifetimes and intensities of dyes with different modes of binding to DNA.
AuthorsSailer BL, Nastasi AJ, Valdez JG, Steinkamp JA, Crissman HA
JournalJ Histochem Cytochem
PubMed ID9016307
'Deuterium oxide (D2O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of ... More
Environment-sensitive labels in multiplex fluorescence analyses of protein-DNA complexes.
AuthorsDrees BL, Rye HS, Glazer AN, Nelson HC
JournalJ Biol Chem
PubMed ID8943271
'Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in ... More
1H NMR studies of the bis-intercalation of a homodimeric oxazole yellow dye in DNA oligonucleotides.
AuthorsJohansen F, Jacobsen JP
JournalJ Biomol Struct Dyn
PubMed ID9833661
'We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric oxazole yellow dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-( 3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene)-quinoliniu m tetraiodide (YOYO), to oligonucleotides containing the (5'-CTAG-3')2 and the (5'-CCGG-3')2 binding sites in either different oligonucleotides or in the same oligonucleotide. YOYO bis-intercalates strongly in all the ... More
Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism.
AuthorsAnterola AM, Jeon JH, Davin LB, Lewis NG
JournalJ Biol Chem
PubMed ID11891223
'Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose ... More
Flow cytometric assessment of viability of lactic acid bacteria.
AuthorsBunthof CJ, Bloemen K, Breeuwer P, Rombouts FM, Abee T
JournalAppl Environ Microbiol
PubMed ID11319119
'The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 ... More
Monitoring phytoplankton, bacterioplankton, and virioplankton in a coastal inlet (Bedford Basin) by flow cytometry.
AuthorsLi WK, Dickie PM
JournalCytometry
PubMed ID11429774
'BACKGROUND: To establish the prevailing state of the ecosystem for the assessment of long-term change, the abundance of microbial plankton in Bedford Basin (Nova Scotia, Canada) is monitored weekly by flow cytometry. METHODS: Phytoplankton are detected by their chlorophyll autofluorescence. Those that contain phycoerythrin are designated as Synechococcus cyanobacteria or ... More
Dynamic bis-intercalation of a homodimeric thiazole orange dye in DNA: evidence of intercalator creeping.
'We have used one and two dimensional exchange 1H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1''-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis- 4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinol inium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)2 (1) and d(CGCTAGCTAGCG)2 (2). TOTO binds preferentially to the ... More
Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.
AuthorsRye HS, Yue S, Wemmer DE, Quesada MA, Haugland RP, Mathies RA, Glazer AN
JournalNucleic Acids Res
PubMed ID1614866
'The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1''-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually ... More
X-ray induced DNA double-strand breaks in human sperm.
AuthorsSingh NP, Stephens RE
JournalMutagenesis
PubMed ID9491398
'A methodology for quantifying DNA double-strand breaks in human sperm is described. Sperm from three healthy human donors on three separate days each were irradiated with 12.5, 25, 50 and 100 cGy X-rays. Linear dose-response effects were observed in migrated DNA from sperm nuclei when electrophoresed under neutral conditions. RNase ... More
High-throughput flow cytometric DNA fragment sizing.
AuthorsVan Orden A, Keller RA, Ambrose WP
JournalAnal Chem
PubMed ID10655632
'The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel ... More
High-sensitivity capillary electrophoresis of double-stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes.
AuthorsZhu H, Clark SM, Benson SC, Rye HS, Glazer AN, Mathies RA
JournalAnal Chem
PubMed ID8067520
'Fluorescence-detected capillary electrophoresis separations of phi X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium ... More
Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor.
AuthorsRye HS, Drees BL, Nelson HC, Glazer AN
JournalJ Biol Chem
PubMed ID8227088
'The gel mobility-shift assay is an important tool for the study of protein-nucleic acid interactions. High detection sensitivity is typically attained by radioisotopic labeling of the target nucleic acid fragments. A novel fluorescence methodology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole yellow homodimers form stable, ... More
Factors governing the assembly of cationic phospholipid-DNA complexes.
AuthorsKennedy MT, Pozharski EV, Rakhmanova VA, MacDonald RC
JournalBiophys J
PubMed ID10692346
'The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic ... More
Bis-intercalation of homodimeric thiazole orange dye derivatives in DNA.
AuthorsPetersen M, Hamed AA, Pedersen EB, Jacobsen JP
JournalBioconjug Chem
PubMed ID9893966
'The thiazole orange dye 1,1''-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-methyl-2,3-dihydro-2(3H)-benzo-1 ,3-thiazolylidene)methyl]quinolinium tetraiodide (TOTO) binds to double-stranded DNA (dsDNA) in a sequence selective bis-intercalation. We have examined the binding of derivatives of TOTO with different substituents on the benzothiazole ring. The analogues are the following: 1,1''-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-[4-[3-(benzyl-2, 3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]-[4-[3-(++ +methy l-2, 3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]tetraio dide (TOTOBzl) and 1,1''-(4,4,8,8-tetramethyl-4, ... More
Concerted intercalation and minor groove recognition of DNA by a homodimeric thiazole orange dye.
'The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5''-CpT-3''):(5''-ApG-3'') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA ... More
Polarization of scatter and fluorescence signals in flow cytometry.
AuthorsAsbury CL, Uy JL, van den Engh G
JournalCytometry
PubMed ID10805928
'BACKGROUND: The pulses of light scatter and fluorescence measured in flow cytometers exhibit varying degrees of polarization. Flow cytometers are heterogeneously sensitive to this polarization, depending on the light source(s), the optical layout, and the types of mirrors and filters used. Therefore, fluorescence polarization can affect apparent intensity ratios between ... More
Spectral measurements of intercalated PCR-amplified short tandem repeat alleles.
AuthorsMarino MA, Devaney JM, Davis PA, Smith JK, Girard JE
JournalAnal Chem
PubMed ID9823710
'Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a ... More
Bisintercalation of homodimeric thiazole orange dyes in DNA: effect of modifying the linker.
'The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis[4-[3-methy l-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO) binds to double-stranded DNA (dsDNA) in a sequence selective bisintercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans over two base pairs in the minor groove. The binding of analogs of TOTO in ... More
Reduced survival of lens epithelial cells in the alphaA-crystallin-knockout mouse.
AuthorsXi JH, Bai F, Andley UP
JournalJ Cell Sci
PubMed ID12584250
'alphaA-Crystallin (alphaA) is a molecular chaperone expressed preferentially in the lens. alphaA transcripts are first detected during the early stages of lens development and its synthesis continues as the lens grows throughout life. alphaA(-/-) mouse lenses are smaller than controls, and lens epithelial cells derived from these mice have diminished ... More
Interaction of dimeric intercalating dyes with single-stranded DNA.
AuthorsRye HS, Glazer AN
JournalNucleic Acids Res
PubMed ID7739900
'The unsymmetrical cyanine dye thiazole orange homodimer (TOTO) binds to single-stranded DNA (ssDNA, M13mp18 ssDNA) to form a fluorescent complex that is stable under the standard conditions of electrophoresis. The stability of this complex is indistinguishable from that of the corresponding complex of TOTO with double-stranded DNA (dsDNA). To examine ... More
Internal structure and dynamics of isolated Escherichia coli nucleoids assessed by fluorescence correlation spectroscopy.
AuthorsRomantsov T, Fishov I, Krichevsky O
JournalBiophys J
PubMed ID17259281
'The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural ... More
TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry.
AuthorsHirons GT, Fawcett JJ, Crissman HA
JournalCytometry
PubMed ID7513274
'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 ... More
Bacterial fingerprinting by flow cytometry: bacterial species discrimination.
AuthorsKim Y, Jett JH, Larson EJ, Penttila JR, Marrone BL, Keller RA
JournalCytometry
PubMed ID10404148
'BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation ... More
Hyperproliferation and p53 status of lens epithelial cells derived from alphaB-crystallin knockout mice.
AuthorsBai F, Xi JH, Wawrousek EF, Fleming TP, Andley UP
JournalJ Biol Chem
PubMed ID12826669
'alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration ... More
Connexin 47 (Cx47)-deficient mice with enhanced green fluorescent protein reporter gene reveal predominant oligodendrocytic expression of Cx47 and display vacuolized myelin in the CNS.
'To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no ... More
Selection of fluorescent probes for flow cytometric viability assessment of Listeria monocytogenes exposed to membrane-active and oxidizing disinfectants.
AuthorsLuppens SB, Barbaras B, Breeuwer P, Rombouts FM, Abee T
JournalJ Food Prot
PubMed ID12929825
'The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action. A further aim of this study was to determine whether ... More
Orientation of the chromophore dipoles in the TOTO-DNA system.
AuthorsSchins JM, Agronskaia A, de Grooth BG, Greve J
JournalCytometry
PubMed ID10520204
'BACKGROUND:Flow cytometry has been applied successfully to the sizing of medium to large-sized DNA molecules, thanks to the excellent staining properties of cyanine chromophores such as TOTO (homodimer of thiazole orange) (Petty et al.: Anal Chem 67:1755-1761, 1995). The hydrodynamic flow, used to focus the sample molecules in a small ... More
Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA.
AuthorsHertoghs KM, Ellis JH, Catchpole IR
JournalNucleic Acids Res
PubMed ID14530430
'The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing ... More
Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport.
AuthorsNoueiry AO, Lucas WJ, Gilbertson RL
JournalCell
PubMed ID8124726
'Plant viruses establish a systemic infection by moving through plasmodesmata, but little is known of the mechanism(s) involved. The roles of two movement-associated proteins of a single-stranded DNA virus were investigated in vivo, using functional proteins expressed in E. coli and microinjection into plant cells. We report here that the ... More
A fluorometric microplate-based assay of submicrogram monomeric actin by inhibition of deoxyribonuclease I.
AuthorsHuang Z, Yue S, You W, Haugland RP
JournalAnal Biochem
PubMed ID8250234
'Inhibition of bovine pancreatic deoxyribonuclease I (DNase I) has been a standard method for specific quantitation of monomeric (G-) actin. The aim of this work is to substantially enhance the sensitivity of this type of G-actin assay by using a fluorescent dye, thiazole orange (TO), which has a high optical ... More
Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons.
AuthorsTaylor AL, Hewett SJ
JournalJ Biol Chem
PubMed ID12235140
'Brain cells in situ contain low concentrations of free polyunsaturated fatty acids such as arachidonic acid (AA) that are released following pathological insults. As a large rise in extracellular [K(+)] accompanies cerebral ischemia, we explored whether this was a stimulus for cellular AA release employing a murine mixed cortical cell ... More
Selective in vivo inhibition of mitogen-activated protein kinase activation using cell-permeable peptides.
AuthorsKelemen BR, Hsiao K, Goueli SA
JournalJ Biol Chem
PubMed ID11756441
'The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinases (MAPKs), is essential for cellular proliferation and differentiation, and thus there exists great interest to develop specific and selective inhibitors of this enzyme. Whereas small molecule inhibitors PD098095 and U0126 have been used to study MAPK/ERK kinase (MEK), ... More
Single-molecule fluorescence spectroscopy of TOTO on poly-AT and poly-GC DNA.
AuthorsBowen BP, Enderlein J, Woodbury NW
JournalPhotochem Photobiol
PubMed ID14743865
'Excited state lifetime and amplitude measurements were made on thiazole orange dimer (TOTO), a dimeric DNA-intercalating fluorophore, at single-molecule concentrations. As expected from previous study, the excited state lifetime of TOTO intercalated in DNA is dependent on the sequence of the double-stranded DNA, having values of 2.2 ns in poly-GC ... More
Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection.
AuthorsGlazer AN, Rye HS
JournalNature
PubMed ID1436062
'Fluorescent intercalation complexes of certain polycationic ligands with double-stranded DNA provide a new class of multichromophore labels for fluorescence assays.'
Cleavage of stretched DNA on glass by histones observed with fluorescence microscopy.
AuthorsTakeda Y, Mafuné F, Kondow T
JournalNucleic Acids Res Suppl
PubMed ID14510443
'A stretched single lambda DNA molecule labeled with TOTO-1 dye molecules was immobilized on a cover slip by casting a solution of DNA-lipid polyion complex containing TOTO-1 dye, and were loaded with an aqueous solution of histone. Fluorescence microscopy applied to the single DNA showed that (1) the TOTO-1 dye ... More
Stretching of a single tethered polymer in a uniform flow.
AuthorsPerkins TT, Smith DE, Larson RG, Chu S
JournalScience
PubMed ID7701345
'The stretching of single, tethered DNA molecules by a flow was directly visualized with fluorescence microscopy. Molecules ranging in length (L) from 22 to 84 micrometers were held stationary against the flow by the optical trapping of a latex microsphere attached to one end. The fractional extension x/L is a ... More
Why molecules move along a temperature gradient.
AuthorsDuhr S, Braun D
JournalProc Natl Acad Sci U S A
PubMed ID17164337
'Molecules drift along temperature gradients, an effect called thermophoresis, the Soret effect, or thermodiffusion. In liquids, its theoretical foundation is the subject of a long-standing debate. By using an all-optical microfluidic fluorescence method, we present experimental results for DNA and polystyrene beads over a large range of particle sizes, salt ... More
P2X7 receptor activates multiple selective dye-permeation pathways in RAW 264.7 and human embryonic kidney 293 cells.
AuthorsCankurtaran-Sayar S, Sayar K, Ugur M,
JournalMol Pharmacol
PubMed ID19749088
'P2X7 receptor has gained an increasing importance as a drug target. One important response to P2X7 receptor stimulation is the uptake of large molecular weight tracers into cells. However, mechanism for this response is not understood clearly, but it is generally believed that a nonselective large pore protein forms this ... More
Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.
AuthorsGoodwin PM, Johnson ME, Martin JC, Ambrose WP, Marrone BL, Jett JH, Keller RA
JournalNucleic Acids Res
PubMed ID8451182
'Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes ... More
Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters.
AuthorsBunthof CJ, Abee T
JournalAppl Environ Microbiol
PubMed ID12039752
'Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 [1''-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3''-trimethylammoniumpropyl)-pyridinium tetraiodide] for (stressed) lactic acid bacteria (C. ... More
Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.
AuthorsNguyen L, Lucas WJ, Ding B, Zaitlin M
JournalProc Natl Acad Sci U S A
PubMed ID8901636
'Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants. In ... More
Characteristics of different nucleic acid staining dyes for DNA fragment sizing by flow cytometry.
AuthorsYan X, Grace WK, Yoshida TM, Habbersett RC, Velappan N, Jett JH, Keller RA, Marrone BL
JournalAnal Chem
PubMed ID10624155
'An efficient and reliable double-stranded DNA (dsDNA) staining protocol for DNA fragment sizing by flow cytometry is presented. The protocol employs 0.8 microM of PicoGreen to label a wide range of DNA concentrations (0.5 ng/mL to 10,000 ng/mL) without regard to the solution dye/bp ratios and without initial quantification of ... More
In situ screening assay for cell viability using a dimeric cyanine nucleic acid stain.
AuthorsBecker B, Clapper J, Harkins KR, Olson JA
JournalAnal Biochem
PubMed ID7527190
'A rapid and sensitive assay is described for the determination of cell viability of adherent and nonadherent cells that can be performed in situ in 96-well microtiter plates using fluorescence plate scanners. The assay, based on dye exclusion, utilizes a plasma membrane-impermeable, dimeric cyanine dye (YOYO-1). YOYO-1 fluoresces brightly only ... More
The human immunodeficiency virus-1 Tat protein activates human umbilical vein endothelial cell E-selectin expression via an NF-kappa B-dependent mechanism.
'Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein ... More
Energy-transfer fluorescent reagents for DNA analyses.
AuthorsGlazer AN, Mathies RA
JournalCurr Opin Biotechnol
PubMed ID9013654
'Fluorescence resonance energy transfer has facilitated the development of a new class of high-performance fluorescent labeling reagents for multiplex analyses of nucleic acids. The enhanced emission of energy transfer (ET) primers has provided a decadic improvement in the performance of automated DNA sequencers. The emission spectral purity of ET primers ... More
Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogram sensitivity.
AuthorsRye HS, Dabora JM, Quesada MA, Mathies RA, Glazer AN
JournalAnal Biochem
PubMed ID7679561
'Thiazole orange homodimer (TOTO; 1,1''-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methy l-2,3- dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow homodimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) bind with very high affinity to nucleic acids with more than a 1000-fold fluorescence enhancement upon binding. A linear dependence of fluorescence intensity on DNA ... More
Spatial and temporal imaging of gas phase protein and DNA produced by matrix-assisted laser desorption.
AuthorsHeise TW, Yeung ES
JournalAnal Chem
PubMed ID8135376
'The sensitivity of a transient imaging technique based on laser-excited fluorescence is shown to be within the levels required for studying matrix-assisted laser-desorption (MALD) of large biomolecules. Results show that film morphology, particularly film thickness, has a major influence on plume dynamics. Fluorescent labeling of protein and of DNA is ... More
Characterization of mitochondrial DNA using low-stringency single specific primer amplification analyzed by laser induced fluorescence--capillary electrophoresis.
AuthorsMarino MA, Weaver KR, Tully LA, Girard JE, Belgrader P
JournalElectrophoresis
PubMed ID8905267
'Polymerase chain reaction (PCR)-based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low-stringency sequence-specific PCR (LSSP-PCR) is an example of a recent method that does not require ... More
Use of thiazole orange homodimer as an alternative to ethidium bromide for DNA detection in agarose gels.
AuthorsWilke WW, Heller MJ, Iakoubova OK, Robinson RA
JournalMod Pathol
PubMed ID8058712
'Detection of polymerase chain reaction-amplified DNA fragments is commonly accomplished by visualizing the products in electrophoretic agarose beds with the use of ethidium bromide under ultraviolet light. However, ethidium bromide is mutagenic, and special handling and disposal precautions must be used. We report the use of a nonmutagenic dye, thiazole ... More
FRET studies of the interaction of dimeric cyanine dyes with DNA.
AuthorsLaib S, Seeger S
JournalJ Fluoresc
PubMed ID15615044
'Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand ... More
Flow microsphere immunoassay-based method of virus quantitation.
AuthorsSamoylova TI, Smith BF
JournalBiotechniques
PubMed ID10457844
'A sensitive assay for adenovirus quantitation in vitro was developed using the flow microsphere immunoassay (FMIA) approach. Polystyrene microspheres were covalently coated with purified anti-adenoviral antibodies and incubated with virus-containing samples. After incubation, the samples were stained with DNA-specific fluorescent dyes, and microsphere-associated fluorescence was quantitated with a flow cytometer. ... More
Staining of DNA in live and fixed cells.
AuthorsCrissman HA, Hirons GT
JournalMethods Cell Biol
PubMed ID7532262
The nucleic acid ligand. A new tool for molecular recognition.
AuthorsMcGown LB, Joseph MJ, Pitner JB, Vonk GP, Linn CP
JournalAnal Chem
PubMed ID8633764
Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry.
AuthorsGuindulain T, Comas J, Vives-Rego J
JournalAppl Environ Microbiol
PubMed ID9361447
Three nucleic acid dyes (SYTO-13, TOTO-1, and YOYO-1) were tested on cultures of Escherichia coli and marine prokaryote populations. These dyes stain the RNA and DNA in E. coli but only respond to DNA in marine populations, according to the histograms obtained after DNase and RNase treatments. ... More
Alternative labeling techniques for automated fluorescence based analysis of PCR products.
AuthorsMansfield ES, Kronick MN
JournalBiotechniques
PubMed ID8373595
We present here convenient and simple methods that utilize two new fluorescent tags, TOTO-1 and fluorescein-12-dUTP, in conjunction with analysis of PCR products on automated, fluorescence-based electrophoretic instruments. These methods should prove valuable in those applications wherein the effort or expense of covalently attaching a fluorescent tag onto one or ... More
Electrodeless dielectrophoresis of single- and double-stranded DNA.
AuthorsChou CF, Tegenfeldt JO, Bakajin O, Chan SS, Cox EC, Darnton N, Duke T, Austin RH
JournalBiophys J
PubMed ID12324434
Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used ... More
From the Cover: The dynamics of genomic-length DNA molecules in 100-nm channels.
AuthorsTegenfeldt JO, Prinz C, Cao H, Chou S, Reisner WW, Riehn R, Wang YM, Cox EC, Sturm JC, Silberzan P, Austin RH
JournalProc Natl Acad Sci U S A
PubMed ID15252203
We show that genomic-length DNA molecules imaged in nanochannels have an extension along the channel that scales linearly with the contour length of the polymer, in agreement with the scaling arguments developed by de Gennes for self-avoiding confined polymers. This fundamental relationship allows us to measure directly the contour length ... More
Direct visualisation of plasmid DNA in individual chromatography adsorbent particles by confocal scanning laser microscopy.
AuthorsLjunglöf A, Bergvall P, Bhikhabhai R, Hjorth R
JournalJ Chromatogr A
PubMed ID10399329
Confocal microscopy was used for the measurement of plasmid DNA adsorbed to individual adsorbent particles intended for anion-exchange and triple helix affinity chromatography. Plasmid DNA was visualized with the fluorescent dye YOYO-1, that forms a highly fluorescent complex with double stranded DNA. Confocal images were translated into fluorescence intensity profiles ... More
Use of stable dye-DNA intercalating complexes to detect cystic fibrosis mutations.
AuthorsAxton RA, Brock DJ
JournalMol Cell Probes
PubMed ID7969200
Several dyes are now known which intercalate into double-stranded DNA with fluorescence enhancement. One such is TOTO (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl-2,3- dihydro-(benzo-1,3-thiazole)-2-methlyidene]-quinolinium tetraiodide), a homodimer of thiazole orange, whose complexes with DNA are stable to electrophoresis and show a 1000-fold enhancement after binding. We demonstrate that this reagent can be successfully used ... More
Enhanced detection of PCR products through use of TOTO and YOYO intercalating dyes with laser induced fluorescence--capillary electrophoresis.
AuthorsSrinivasan K, Morris SC, Girard JE, Kline MC, Reeder DJ
JournalAppl Theor Electrophor
PubMed ID8218477
Recent developments in the chemical synthesis of DNA-binding dyes have enhanced detection of polymerase chain reaction (PCR) products by capillary electrophoresis. These dyes are dimers of thiazole orange (TOTO) or oxazole orange (YOYO) and have a very high binding affinity for DNA (Haugland, 1992). These dyes show enhanced fluorescence signals ... More
Brownian motion of DNA confined within a two-dimensional array.
AuthorsNykypanchuk D, Strey HH, Hoagland DA
JournalScience
PubMed ID12169727
Linear DNA molecules are visualized while undergoing Brownian motion inside media patterned with molecular-sized spatial constraints. The media, prepared by colloidal templating, trap the macromolecules within a two-dimensional array of spherical cavities interconnected by circular holes. Across a broad DNA size range, diffusion does not proceed by the familiar mechanisms ... More
DNA staining for fluorescence and laser confocal microscopy.
AuthorsSuzuki T, Fujikura K, Higashiyama T, Takata K
JournalJ Histochem Cytochem
PubMed ID9010468
We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, ... More
The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules.
AuthorsUy JL, Asbury CL, Petersen TW, van den Engh G,
JournalCytometry A
PubMed ID15351985
BACKGROUND: The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two ... More
Flow-cytometric analysis of mouse embryonic stem cell lipofection using small and large DNA constructs.
AuthorsMcLenachan S, Sarsero JP, Ioannou PA
JournalGenomics
PubMed ID17449222
Using the lipofection reagent LipofectAMINE 2000 we have examined the delivery of plasmid DNA (5-200 kb) to mouse embryonic stem (mES) cells by flow cytometry. To follow the physical uptake of lipoplexes we labeled DNA molecules with the fluorescent dye TOTO-1. In parallel, expression of an EGFP reporter cassette in ... More
Sizing of single fluorescently stained DNA fragments by scanning microscopy.
AuthorsLaib S, Rankl M, Ruckstuhl T, Seeger S
JournalNucleic Acids Res
PubMed ID14602931
We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed ... More
Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis.
AuthorsShi Y, Simpson PC, Scherer JR, Wexler D, Skibola C, Smith MT, Mathies RA
JournalAnal Chem
PubMed ID10596215
The design, fabrication, and operation of a radial capillary array electrophoresis microplate and scanner for high-throughput DNA analysis is presented. The microplate consists of a central common anode reservoir coupled to 96 separate microfabricated separation channels connected to sample injectors on the perimeter of the 10-cm-diameter wafer. Detection is accomplished ... More
Transport of nucleosome core particles in semidilute DNA solutions.
AuthorsMangenot S, Keller S, Rädler J
JournalBiophys J
PubMed ID12944295
We studied the diffusion of native and trypsinized nucleosome core particles (NCPs), in aqueous solution and in concentrated DNA solutions (0.25-100 mg/ml) using fluorescence correlation spectroscopy (FCS). The highest DNA concentrations studied mimic the DNA density inside the cell nucleus. The diffusion coefficient of freely diffusing NCPs depends on the ... More
Homogeneous fluorescence detection method for human leukocyte antigen-DR typing following polymerase chain reaction amplification with sequence-specific primer.
AuthorsOkamoto N, Lee A, Kano T, Lee TD
JournalAnal Biochem
PubMed ID7810876
A fluorescent homogeneous method for the detection of sequence-specific amplification of human leukocyte antigen (HLA) alleles has been developed. In this approach, polymerase chain reaction sequence-specific primers (PCR-SSP) are used to amplify DRB1, DRB3, and DRB4 alleles. Lambda exonuclease and Exonuclease I are added to reduce background by digesting template ... More
High-speed parallel separation of DNA restriction fragments using capillary array electrophoresis.
AuthorsClark SM, Mathies RA
JournalAnal Biochem
PubMed ID8122774
A new method for performing high-speed, high-throughput sizing of DNA has been developed. Samples containing DNA restriction fragments between 70 and 10,000 base pairs in length are electrophoretically separated using capillary arrays in approximately 20 min and detected with high sensitivity using a fluorescence detection system. The separations of phi ... More
DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags.
AuthorsChan EY, Goncalves NM, Haeusler RA, Hatch AJ, Larson JW, Maletta AM, Yantz GR, Carstea ED, Fuchs M, Wong GG, Gullans SR, Gilmanshin R
JournalGenome Res
PubMed ID15173119
We have developed a rapid molecular mapping technology--Direct Linear Analysis (DLA)--on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. ... More
Double bands in DNA gel electrophoresis caused by bis-intercalating dyes.
AuthorsCarlsson C, Jonsson M, Akerman B
JournalNucleic Acids Res
PubMed ID7630719
Many bis-intercalating dyes used for fluorescence detection of DNA in electrophoresis have been reported to give band-splitting and band-broadening, which results in poor resolution and a decreased detection sensitivity. We have studied the dimeric dye YOYO-1, and to some extent also TOTO-1 and EthD-1, and found that in complex with ... More
Characterization of regions in hsMAD1 needed for binding hsMAD2. A polymorphic change in an hsMAD1 leucine zipper affects MAD1-MAD2 interaction and spindle checkpoint function.
In eukaryotes, the mitotic spindle assembly checkpoint provides a monitor for the fidelity of chromosomal segregation. In this context, the mitotic arrest deficiency protein 2 (MAD2) censors chromosomal mis-segregation by monitoring microtubule attachment/tension, a role that requires its attachment to kinetochores. Studies in yeast have shown that binding of MAD1 ... More
Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides.
AuthorsJacobsen JP, Pedersen JB, Hansen LF, Wemmer DE
JournalNucleic Acids Res
PubMed ID7708489
We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4- (3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolin ium tetraiodide (TOTO), to various double stranded DNA oligonucleotides. TOTO binds strongly to all the oligonucleotides used, but usually more than one complex is observed and exchange between ... More
Purkinje cell dysfunction and alteration of long-term synaptic plasticity in fetal alcohol syndrome.
AuthorsServais L, Hourez R, Bearzatto B, Gall D, Schiffmann SN, Cheron G
JournalProc Natl Acad Sci U S A
PubMed ID17535929
In cerebellum and other brain regions, neuronal cell death because of ethanol consumption by the mother is thought to be the leading cause of neurological deficits in the offspring. However, little is known about how surviving cells function. We studied cerebellar Purkinje cells in vivo and in vitro to determine ... More
Confocal DNA cytometry: a contour-based segmentation algorithm for automated three-dimensional image segmentation.
AuthorsBeliën JA, van Ginkel HA, Tekola P, Ploeger LS, Poulin NM, Baak JP, van Diest PJ
JournalCytometry
PubMed ID12210606
BACKGROUND: Confocal laser scanning microscopy (CLSM) presents the opportunity to perform three-dimensional (3D) DNA content measurements on intact cells in thick histological sections. So far, these measurements have been performed manually, which is quite time-consuming. METHODS: In this study, an intuitive contour-based segmentation algorithm for automatic 3D CLSM image cytometry ... More
Direct visualization at the single-cell level of electrically mediated gene delivery.
AuthorsGolzio M, Teissie J, Rols MP
JournalProc Natl Acad Sci U S A
PubMed ID11818537
Electropermeabilization is one of the nonviral methods successfully used to transfer genes into living cells in vitro and in vivo. Although this approach shows promise in the field of gene therapy, very little is known about the basic processes supporting DNA transfer. The present investigation studies this process at the ... More
Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis.
AuthorsMurphy FR, Issa R, Zhou X, Ratnarajah S, Nagase H, Arthur MJ, Benyon C, Iredale JP
JournalJ Biol Chem
PubMed ID11796725
The activated hepatic stellate cell (HSC) is central to liver fibrosis as the major source of collagens I and III and the tissue inhibitors of metalloproteinase-1 (TIMP-1). During spontaneous recovery from liver fibrosis, there is a decrease of TIMP expression, an increase in collagenase activity, and increased apoptosis of HSC, ... More
Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA.
AuthorsLiu H, Boulton MI, Thomas CL, Prior DA, Oparka KJ, Davies JW
JournalMol Plant Microbe Interact
PubMed ID10517029
Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the ... More
Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells.
We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward ... More
Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis.
AuthorsSonehara T, Kojima K, Irie T
JournalAnal Chem
PubMed ID12380839
Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary ... More
Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation.
AuthorsBelecky-Adams TL, Adler R, Beebe DC
JournalDevelopment
PubMed ID12135918
Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the ... More
The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin.
Authors Wilkinson Shane R; Meyer David J; Taylor Martin C; Bromley Elizabeth V; Miles Michael A; Kelly John M;
JournalJ Biol Chem
PubMed ID11842085
Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction ... More
SW13 cells can transition between two distinct subtypes by switching expression of BRG1 and Brm genes at the post-transcriptional level.
AuthorsYamamichi-Nishina M, Ito T, Mizutani T, Yamamichi N, Watanabe H, Iba H
JournalJ Biol Chem
PubMed ID12493776
The human adrenal carcinoma cell line, SW13, has been reported to be deficient in both BRG1 and Brm expression and therefore is considered to lack a functional SWI/SNF complex. We found that the original cell line of SW13 is composed of two subtypes, one that expresses neither BRG1 nor Brm ... More
On the sequence selective bis-intercalation of a homodimeric thiazole orange dye in DNA.
AuthorsBunkenborg J, Stidsen MM, Jacobsen JP
JournalBioconjug Chem
PubMed ID10502349
The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-methyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTO) binds sequence selectively to double-stranded DNA (dsDNA) by bis-intercalation. Each chromophore is sandwiched between two base pairs in a d(5'-py-p-py-3'):d(5'-pu-p-pu-3') site, and the linker spans over two base pairs in the minor groove. We have examined the binding of TOTO to ... More
Polymorphisms in the human high sulfur hair keratin-associated protein 1, KAP1, gene family.
AuthorsShimomura Y, Aoki N, Schweizer J, Langbein L, Rogers MA, Winter H, Ito M
JournalJ Biol Chem
PubMed ID12228244
Hair fiber differentiation and maturation involves the close interaction between hair keratins and their associated proteins, KAPs. Recently, a cluster of seven human KAP multigen families has been identified on chromosome 17q12-21 among which were four hKAP1 genes (hKAP1.1B, hKAP1.3, hKAP1.4, and hKAP1.5). In addition, there were previous as well ... More
Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.
AuthorsMansfield ES, Robertson JM, Lebo RV, Lucero MY, Mayrand PE, Rappaport E, Parrella T, Sartore M, Surrey S, Fortina P
JournalAm J Med Genet
PubMed ID7510932
Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR ... More
Bacteria genome fingerprinting by flow cytometry.
AuthorsHuang Z, Jett JH, Keller RA
JournalCytometry
PubMed ID10554173
BACKGROUND: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus ... More
Megalin functions as an endocytic sonic hedgehog receptor.
Embryos deficient in the morphogen Sonic hedgehog (Shh) or the endocytic receptor megalin exhibit common neurodevelopmental abnormalities. Therefore, we have investigated the possibility that a functional relationship exists between the two proteins. During embryonic development, megalin was found to be expressed along the apical surfaces of neuroepithelial cells and was ... More
Cytokine mRNA levels in unmanipulated (ex vivo) and in vitro stimulated monkey PBMCs using a semi-quantitative RT-PCR and high sensitivity fluorescence-based detection strategy.
AuthorsBenveniste O, Vaslin B, Villinger F, Le Grand R, Ansari AA, Dormont D
JournalCytokine
PubMed ID8742064
To investigate the spectrum of cytokines expressed by peripheral blood mononuclear cells (PBMC) from cynomolgus macaques (Macaca fascicularis), we used a semi-quantitative RT-PCR to determine levels of mRNA coding for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. The PCR products were labelled and quantified using a new fluorescent ... More
Bis-intercalation of a homodimeric thiazole orange dye in DNA in symmetrical pyrimidine-pyrimidine-purine-purine oligonucleotides.
AuthorsHansen LF, Jensen LK, Jacobsen JP
JournalNucleic Acids Res
PubMed ID8600453
One- and two-dimensional 1H NMR spectroscopy were used to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-(3 -methyl-2,3-dihydro-(benzo- 1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to various double-stranded DNA oligonucleotides containing symmetric (5'-pyr-pyr-pu-pu-3')2 or (5'-pu-pu-pyr-pyr-3')2 sequences. It was found that TOTO binds preferentially to oligonucleotides containing a (5'-CTAG-3')2 or a (5'-CCGG-3')2 sequence. ... More
Dynamics of a bis-intercalator DNA complex by 1H-detected natural abundance 13C NMR spectroscopy.
AuthorsSpielmann HP
JournalBiochemistry
PubMed ID9836579
The dynamics of the DNA oligomer d(CGCTAGCG)2 (CTSYM) and its complex with the dye 1,1-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1, 3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO) (CTSYMTOTO) bis-intercalated at the 5'-CT-3' sequence steps have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data were acquired and have been analyzed ... More
Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.
AuthorsSuzuma I, Suzuma K, Ueki K, Hata Y, Feener EP, King GL, Aiello LP
JournalJ Biol Chem
PubMed ID11694503
Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by ... More