Switch to anaerobic glucose metabolism with NADH accumulation in the beta-cell model of mitochondrial diabetes. Characteristics of betaHC9 cells deficient in mitochondrial DNA transcription.
To elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 microg/ml ethidium bromide (EtBr) to the murine pancreatic beta cell line betaHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of ... More
Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.
AuthorsDrevets DA, Campbell PA
JournalJ Immunol Methods
PubMed ID1919019
One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et ... More
Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.
AuthorsLevashina EA, Moita LF, Blandin S, Vriend G, Lagueux M, Kafatos FC
JournalCell
PubMed ID11257225
'We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as ... More
Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization.
AuthorsMazzoni A, Young HA, Spitzer JH, Visintin A, Segal DM
JournalJ Clin Invest
PubMed ID11748270
'Atopic diseases such as allergy and asthma are characterized by increases in Th2 cells and serum IgE antibodies. The binding of allergens to IgE on mast cells triggers the release of several mediators, of which histamine is the most prevalent. Here we show that histamine, together with a maturation signal, ... More
Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses.
AuthorsItoh S, Thorikay M, Kowanetz M, Moustakas A, Itoh F, Heldin CH, ten Dijke P
JournalJ Biol Chem
PubMed ID12446693
'Transforming growth factor-beta (TGF-beta) elicits cellular effects by activating specific Smad proteins that control the transcription of target genes. Whereas there is growing evidence that there are TGF-beta type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-beta signaling is not well ... More
A kinase-independent function of Ask1 in caspase-independent cell death.
AuthorsCharette SJ, Lambert H, Landry J
JournalJ Biol Chem
PubMed ID11493600
'Ask1 (apoptosis signal-regulating kinase 1) is activated as a consequence of cell exposure to a variety of stresses and can then initiate apoptosis. A known pathway of apoptosis downstream of Ask1 involves the activation of the stress-activated protein kinases, the release of cytochrome c from mitochondria, the activation of caspases, ... More
Specific recruitment of human cohesin to laser-induced DNA damage.
AuthorsKim JS, Krasieva TB, LaMorte V, Taylor AM, Yokomori K
JournalJ Biol Chem
PubMed ID12228239
'Cohesin is a conserved multiprotein complex that plays an essential role in sister chromatid cohesion. During interphase, cohesin is required for the establishment of cohesion following DNA replication. Because cohesin mutants resulted in increased sensitivity to DNA damage, a role for cohesin in DNA repair was also suggested. However, it ... More
IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines.
AuthorsAlbanesi C, Scarponi C, Sebastiani S, Cavani A, Federici M, De Pità O, Puddu P, Girolomoni G
JournalJ Immunol
PubMed ID10903743
'IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, ... More
High-resolution detection of loss of heterozygosity of dinucleotide microsatellite markers.
AuthorsHourihan RN, O'Sullivan GC, Morgan JG
JournalBiotechniques
PubMed ID11233603
'Dinucleotide microsatellite markers are frequently investigated to study inheritance, genetic stability, and allele frequency distribution in a wide variety of genetic disorders. Previous studies have encountered significant problems regarding resolution and detection of dinucleotide, microsatellites. In this study, a useful method to investigate loss of heterozygosity (LOH) of dinucleotide microsatellite ... More
Chronic hypoxia protects against gamma-irradiation-induced apoptosis by inducing bcl-2 up-regulation and inhibiting mitochondrial translocation and conformational change of bax protein.
AuthorsCuisnier O, Serduc R, Lavieille JP, Longuet M, Reyt E, Riva C
JournalInt J Oncol
PubMed ID12963983
'Malignant tumours contain zones of chronic or acute hypoxia, which influence their prognosis and progression. The goal of our study was to understand the role of hypoxia in radio-resistance in a squamous cell carcinoma cell line of the head and neck (KB-3-1 cells). Cell growth was evaluated by Trypan blue ... More
Some fluorescent counterstains for neuroanatomical studies.
AuthorsSchmued LC, Swanson LW, Sawchenko PE
JournalJ Histochem Cytochem
PubMed ID6174560
'Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ... More
A polarized photobleaching study of chromatin reorientation in intact nuclei.
AuthorsSelvin PR, Scalettar BA, Langmore JP, Axelrod D, Klein MP, Hearst JE
JournalJ Mol Biol
PubMed ID2388274
'Polarized fluorescence recovery after photobleaching (pFRAP) was used to monitor the effects that condensation, i.e. compaction and aggregation, have on the (microseconds and ms) internal dynamics of chromatin in intact nuclei. When divalent cations were present with physiological (approximately 90 mM) monovalent salt the chromatin was found to exist in ... More
Patch-clamp detection of macromolecular translocation along nuclear pores.
AuthorsBustamante JO, Varanda WA
JournalBraz J Med Biol Res
PubMed ID9698781
'The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal ''gigaseal'' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability ... More
Connexin26 is responsible for anionic molecule permeability in the cochlea for intercellular signalling and metabolic communications.
AuthorsZhao HB
JournalEur J Neurosci
PubMed ID15869481
'Abstract A gap junction is composed of two hemichannels and possesses a relatively large pore size ( approximately 10-15 A), allowing passage of ions and molecules up to 1 kDa. Here, we report that connexin hemichannels and gap junctions in the guinea pig cochlea had significant charge selectivity among permeating ... More
Fluorimetric quantification of cell death in monolayer cultures and cell suspensions.
AuthorsRuiz MC, Michelangeli F, Ludert JE, Liprandi F, del Castillo JR, Chemello ME, Benaim G, Cohen E
JournalJ Biochem Biophys Methods
PubMed ID1779095
'A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania). Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in ... More
DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.
AuthorsEven-Chen S, Barenholz Y
JournalBiochim Biophys Acta
PubMed ID11118529
'Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to ... More
Fluorescent derivatives of yeast tRNAPhe.
AuthorsWintermeyer W, Zachau HG
JournalEur J Biochem
PubMed ID114393
'The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described. The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4-reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by ... More
Lack of bradykinin-induced smooth muscle cell hyperpolarization despite heterocellular dye coupling and endothelial cell hyperpolarization in porcine ciliary artery.
AuthorsBény JL, Zhu P, Haefliger IO
JournalJ Vasc Res
PubMed ID9349728
'In porcine coronary artery, bradykinin-induced endothelium-dependent vasodilatations are associated with simultaneous endothelium as well as endothelium-dependent smooth muscle cell (SMC) hyperpolarizations. In contrast, in porcine ciliary artery bradykinin evokes endothelium-dependent relaxations, but no change in SMC membrane potential. This study addresses the question of whether the lack of bradykinin-induced SMC ... More
A rapid evaluation of phagocytosis and killing of Candida albicans by CD13+ leukocytes.
AuthorsSaresella M, Roda K, Speciale L, Taramelli D, Mendozzi E, Guerini F, Ferrante P
JournalJ Immunol Methods
PubMed ID9520305
'Flow cytometry can be adopted for routine monitoring of the immune functions of human polymorphonuclear leukocytes (PMNs) in several disease states. We recently developed a rapid and reproducible assay for the evaluation of the phagocytosis and killing of Candida albicans blastospores by human PMNs. Whole blood leukocytes were incubated with ... 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
A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry.
AuthorsSchmid I, Uittenbogaart CH, Keld B, Giorgi JV
JournalJ Immunol Methods
PubMed ID8157993
'A sensitive method for quantification of cells undergoing apoptosis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead ... More
Tip110, the human immunodeficiency virus type 1 (HIV-1) Tat-interacting protein of 110 kDa as a negative regulator of androgen receptor (AR) transcriptional activation.
AuthorsLiu Y, Kim BO, Kao C, Jung C, Dalton JT, He JJ
JournalJ Biol Chem
PubMed ID15031286
'Upon binding to androgen, androgen receptor (AR) can activate expression of target genes through its direct binding to the androgen-responsive elements (AREs), which are located within the target gene promoters and/or enhancers. A number of cellular proteins have been identified as co-regulators to regulate this transactivation process. One common structural ... More
Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde.
AuthorsLarsen JK, Munch-Petersen B, Christiansen J, Jørgensen K
JournalCytometry
PubMed ID2419056
'Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than ... More
Dynamical change of mitochondrial DNA induced in the living cell by perturbing the electrochemical gradient.
AuthorsCoppey-Moisan M, Brunet AC, Morais R, Coppey J
JournalBiophys J
PubMed ID8913573
'Digital-imaging microscopy was used in conditions that allowed the native state to be preserved and hence fluorescence variations of specific probes to be followed in the real time of living mammalian cells. Ethidium bromide was shown to enter into living cells and to intercalate stably into mitochondrial DNA (mtDNA), giving ... More
Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells.
AuthorsTramier M, Kemnitz K, Durieux C, Coppey J, Denjean P, Pansu RB, Coppey-Moisan M
JournalBiophys J
PubMed ID10777758
'Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting ... More
Intercalative interactions of ethidium dyes with triplex structures.
AuthorsTuite E, Nordén B
JournalBioorg Med Chem
PubMed ID7582948
'The binding of phenanthridine dyes to triplex poly(dT)*poly(dA).poly(dT) and its precursor duplex poly(dA).poly(dT) is characterized using linear dichroism and circular dichroism spectroscopy, and thermal denaturation. The two monomeric dyes ethidium bromide and propidium iodide are shown to behave similarly to each other in intercalating into and stabilizing both the duplex ... More
Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level.
AuthorsBiggiogera M, Biggiogera FF
JournalJ Histochem Cytochem
PubMed ID2471727
'Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only ... More
Slit-scan flow cytometry of mammalian chromosomes.
AuthorsGray JW, Peters D, Merrill JT, Martin R, Van Dilla MA
JournalJ Histochem Cytochem
PubMed ID374608
'A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded ... More
Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells.
AuthorsHimpens B, De Smedt H, Droogmans G, Casteels R
JournalAm J Physiol
PubMed ID1386189
'The free Ca2+ concentrations in the nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured smooth muscle cells were estimated using the fluorescent dye indo-1 and the ACAS 570 confocal laser microscope. In resting DDT1MF2 smooth muscle cells [Ca2+]n was found to be lower than [Ca2+]c. Both values increased transiently in response ... 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
Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells.
AuthorsEstacion M, Schilling WP
JournalBMC Physiol
PubMed ID11231888
'BACKGROUND: Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, ... More
Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane.
AuthorsGabriel B, Teissié J
JournalBiophys J
PubMed ID9370457
'Interaction of two stains (propidium iodide and ethidium bromide) with electropermeabilized living Chinese hamster ovary cells is observed using an ultrafast fluorescence image acquisition system. The computing process is linked to an ultra-low-light intensifying camera working with a very short time resolution (3.33 ms per image). Altered parts of the ... More
Fluorescent labels influence phagocytosis of Bordetella pertussis by human neutrophils.
AuthorsWeingart CL, Broitman-Maduro G, Dean G, Newman S, Peppler M, Weiss AA
JournalInfect Immun
PubMed ID10417202
'To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled ... More
Switching on of the proliferation or apoptosis of activated human T lymphocytes by IFN-gamma is correlated with the differential expression of the alpha- and beta-chains of its receptor.
AuthorsNovelli F, Bernabei P, Ozmen L, Rigamonti L, Allione A, Pestka S, Garotta G, Forni G
JournalJ Immunol
PubMed ID8757312
'To find out how physiologically secreted IFN-gamma controls either the proliferation or the apoptosis of human T lymphocytes, the kinetics of expression of the alpha- and beta-chains of its receptor (IFN-gamma R) were sequentially followed on T lymphocytes first activated with PHA and then cultured in the presence of IL-2, ... More
Green-light transilluminator for the detection without photodamage of proteins and DNA labeled with different fluorescent dyes.
AuthorsAlba FJ, Bermúdez A, Daban JR
JournalElectrophoresis
PubMed ID11258745
'The excitation spectra of Nile red and SYPRO red, two currently used dyes for the fluorescent staining of protein bands in sodium dodecyl sulfate (SDS)-polyacrylamide gels, show an excitation peak in the UV region and another in the visible region (maximum at about 550 nm). Ethidium bromide and other intercalating ... 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
Maitotoxin activates a nonselective cation channel and a P2Z/P2X(7)-like cytolytic pore in human skin fibroblasts.
AuthorsSchilling WP, Sinkins WG, Estacion M
JournalAm J Physiol
PubMed ID10516106
'Maitotoxin (MTX), a potent cytolytic agent, activates Ca(2+) entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts ... More
Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.
AuthorsLittle TL, Xia J, Duling BR
JournalCirc Res
PubMed ID7859395
'Dye tracers were chosen, based on net charge, chemical structure, and reactive groups, to test for the existence of and to provide novel insight into channel selectivities of junctional pathways connecting smooth muscle and endothelial cells of the arteriolar wall. Dyes were injected into individual smooth muscle or endothelial cells ... More
Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator.
AuthorsYguerabide J, Ceballos A
JournalAnal Biochem
PubMed ID8572297
'We present a quantitative fluorescence method for continuous measurement of DNA or RNA hybridization (including renaturation) kinetics using a fluorescent DNA intercalator. The method has high sensitivity and can be used with reaction volumes as small as 1 microliter and amounts of DNA around 1 ng. The method is based ... More
Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria.
AuthorsGenini D, Adachi S, Chao Q, Rose DW, Carrera CJ, Cottam HB, Carson DA, Leoni LM
JournalBlood
PubMed ID11071652
'Adenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to ... More
Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils.
AuthorsPerticarari S, Presani G, Mangiarotti MA, Banfi E
JournalCytometry
PubMed ID1782835
'We developed a rapid and sensitive two-color flow cytometric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the ... More
Induction of necrosis by zinc in prostate carcinoma cells and identification of proteins increased in association with this induction.
AuthorsIguchi K, Hamatake M, Ishida R, Usami Y, Adachi T, Yamamoto H, Koshida K, Uchibayashi T, Hirano K
JournalEur J Biochem
PubMed ID9654077
'Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this type of cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostate carcinoma cells. When zinc acetate was added to LNCaP or PC-3 cells in monolayer ... More
Binding of ethidium to the nucleosome core particle. 1. Binding and dissociation reactions.
AuthorsMcMurray CT, van Holde KE
JournalBiochemistry
PubMed ID1904272
'We have examined binding properties of and dissociation induced by the intercalating dye ethidium bromide when it interacts with the nucleosome core particle under low ionic strength conditions. Ethidium binding to the core particle results in a reversible dissociation which requires the critical binding of 14 ethidium molecules. Under low ... More
Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry.
AuthorsTelford WG, King LE, Fraker PJ
JournalCytometry
PubMed ID1372208
'Mouse thymocytes readily undergo apoptosis-associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide-stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A0 region) below the G0/G1 region. Cells in this ... More
Membrane permeabilization by alpha-helical peptides: a flow cytometry study.
AuthorsMidoux P, Mayer R, Monsigny M
JournalBiochim Biophys Acta
PubMed ID7488630
'The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-helical peptides ... More
Manual punch versus power harvesting of osteochondral grafts.
AuthorsEvans PJ, Miniaci A, Hurtig MB
JournalArthroscopy
PubMed ID15007320
'PURPOSE: The purpose of the study was to analyze the effect on chondrocyte viability of 2 existing methods of harvesting osteochondral grafts used for articular cartilage resurfacing. TYPE OF STUDY: Acute animal experiment. METHODS: Power (P) trephine versus manual (M) punch harvesting was tested; 2.7-mm and 4.5-mm dowels were harvested ... More
Mouse microglial cells express a plasma membrane pore gated by extracellular ATP.
AuthorsFerrari D, Villalba M, Chiozzi P, Falzoni S, Ricciardi-Castagnoli P, Di Virgilio F
JournalJ Immunol
PubMed ID8568257
'We have investigated responses to extracellular ATP (ATPe) in the microglial cell lines N9 and N13 and in freshly isolated mouse microglial cells. Upon stimulation with this nucleotide, N9 and N13 cells underwent an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i), a sustained depolarization of the plasma membrane, and ... More
In situ assessment of cell viability.
AuthorsYang H, Acker J, Chen A, McGann L
JournalCell Transplant
PubMed ID9786064
'Cryobiological studies of tissues often require the simultaneous assessment of tissue structure and in situ cellular function. Localization of damage during cryopreservation occurs as a consequence of tissue structure and morphology and as a result of biophysical constraints imposed by diffusion and heat transfer. This study used five experimental model ... More
Brain-derived neurotrophic factor can act as a pronecrotic factor through transcriptional and translational activation of NADPH oxidase.
AuthorsKim SH, Won SJ, Sohn S, Kwon HJ, Lee JY, Park JH, Gwag BJ
JournalJ Cell Biol
PubMed ID12460985
'Several lines of evidence suggest that neurotrophins (NTs) potentiate or cause neuronal injury under various pathological conditions. Since NTs enhance survival and differentiation of cultured neurons in serum or defined media containing antioxidants, we set out experiments to delineate the patterns and underlying mechanisms of brain-derived neurotrophic factor (BDNF)-induced neuronal ... More
Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.
AuthorsRobinson KM, Janes MS, Pehar M, Monette JS, Ross MF, Hagen TM, Murphy MP, Beckman JS
JournalProc Natl Acad Sci U S A
PubMed ID17015830
'The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo ... More
An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence.
AuthorsSeverini A, Morgan AR
JournalAnal Biochem
PubMed ID2042745
'Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their ... More
Bioenergetics of the staphylococcal multidrug export protein QacA. Identification of distinct binding sites for monovalent and divalent cations.
AuthorsMitchell BA, Paulsen IT, Brown MH, Skurray RA
JournalJ Biol Chem
PubMed ID9920900
'The multidrug efflux pump QacA from Staphylococcus aureus confers resistance to an extensive range of structurally dissimilar compounds. Fluorimetric analyses demonstrated that QacA confers resistance to the divalent cation 4'',6-diamidino-2-phenylindole, utilizing a proton motive force-dependent efflux mechanism previously demonstrated for QacA-mediated resistance to the monovalent cation ethidium. Both the ionophores ... More
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
'Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting ... More
Modifications of alkaline microgel electrophoresis for sensitive detection of DNA damage.
AuthorsSingh NP, Stephens RE, Schneider EL
JournalInt J Radiat Biol
PubMed ID8027608
'The alkaline microgel electrophoresis technique was modified to achieve a substantial increase in sensitivity for the detection of radiation-induced DNA damage in human lymphocytes. This increased sensitivity was achieved through: (1) the addition of free radical scavengers to the electrophoresis solution to reduce DNA damage generated during alkaline unwinding and ... More
Hydration changes for DNA intercalation reactions.
AuthorsQu X, Chaires JB
JournalJ Am Chem Soc
PubMed ID11273594
'The hydration changes that accompany the DNA binding of five intercalators (ethidium, propidium, proflavine, daunomycin, and 7-aminoactinomycin D) were measured by the osmotic stress method with use of the osmolytes betaine, sucrose, and triethylene glycol. Water uptake was found to accompany complex formation for all intercalators except ethidium. The difference ... More
Modulation of the viral ATPase activity by the portal protein correlates with DNA packaging efficiency.
AuthorsOliveira L, Henriques AO, Tavares P
JournalJ Biol Chem
PubMed ID16735502
'DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the ... More
Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments.
'DNA quantification of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quantification is impaired by humic acids (HA). Due to its lack of specificity and sensitivity, ... More
Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.
'After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5- (and -6)-carboxy-x-rhodamine], was microinjected into living oocytes. Biotinylated ... More
Interactions between pairs of DNA-binding dyes: results and implications of chromosome analysis.
AuthorsLatt SA, Sahar E, Eisenhard ME, Juergens LA
JournalCytometry
PubMed ID6168450
'A number of DNA-binding dyes, with spectral properties making them suitable as components of energy donor-acceptor pairs, are described. If such pairs are used to stain metaphase chromosomes, and if the energy acceptor (e.g., actinomycin D or methyl green) has a binding specificity opposite to the binding or fluorescence specificity ... More
Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding.
AuthorsKubbies M
JournalCytometry
PubMed ID1692786
'Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X-rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non-cycling cell fraction; and a distinctive, non-cycling G-/G1 subpopulation appears which is characterized by ... More
Calpain activation in apoptosis.
AuthorsSquìer MK, Miller AC, Malkinson AM, Cohen JJ
JournalJ Cell Physiol
PubMed ID8163563
'Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in ... More
Factors affecting quantitation of DNA bands in gels using a charge-coupled device imaging system.
AuthorsWhite HW, Wu M
JournalElectrophoresis
PubMed ID11332753
'Quantitation of DNA bands separated in polyacrylamide or agarose gels was tested under a variety of conditions to examine key factors contributing to the ability to obtain quantitative data. Variations tested included comparison of different fluorescent stains (ethidium bromide and GelStar stain), and variations of other parameters relating to the ... More
Flow cytometry of bacterial cells: comparison between different flow cytometers and different DNA stains.
AuthorsBernander R, Stokke T, Boye E
JournalCytometry
PubMed ID9450522
'The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations. The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers ... More
Pharmacologic properties of P(2Z)/P2X(7 )receptor characterized in murine dendritic cells: role on the induction of apoptosis.
AuthorsNihei OK, de Carvalho AC, Savino W, Alves LA
JournalBlood
PubMed ID10910915
'In the immune system, extracellular adenosine 5''-triphosphate (ATP) mediates a variety of effects mainly through activation of a particular receptor subtype, the pore-forming P(2Z)/P2X(7) purinoceptor. This purinergic receptor has been described chiefly in cells of hemopoietic origin such as T cells, thymocytes, monocytes, macrophages, and phagocytic cells of thymic reticulum. ... More
Probing chromatin structure in the early phases of apoptosis.
AuthorsFerlini C, Biselli R, Scambia G, Fattorossi A
JournalCell Prolif
PubMed ID8883466
'A typical flow cytometric marker of apoptosis is the appearance of a hypodiploid peak. This phenomenon is related to the chromatin fragmentation and loss that occurs during the late stages of the process. We describe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human ... More
Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique.
AuthorsFernandez-Patron C, Castellanos-Serra L, Hardy E, Guerra M, Estevez E, Mehl E, Frank RW
JournalElectrophoresis
PubMed ID9820957
'We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels can be detected by reverse-staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gel-isolated nucleic acids and bacterial glycolipids. Because there is ... More
Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor.
AuthorsPan Z, Damron D, Nieminen AL, Bhat MB, Ma J
JournalJ Biol Chem
PubMed ID10764805
'Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ... More
Differential intracellular labelling of identified neurones with two fluorescent dyes.
AuthorsMcCarthy PW, Lawson SN
JournalBrain Res Bull
PubMed ID3370508
'Electrophysiologically characterised rat dorsal root ganglion (DRG) neurones in vitro were injected with a fluorescent dye, either ethidium bromide or Lucifer yellow. Microelectrodes filled with ethidium bromide had lower resistances and were electrically more stable than comparable electrodes filled with Lucifer yellow. Neither dye affected the electrophysiology of these neurones ... More
Nucleotide receptors: an emerging family of regulatory molecules in blood cells.
AuthorsDi Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torboli M, Bolognesi G, Baricordi OR
JournalBlood
PubMed ID11157473
'Nucleotides are emerging as an ubiquitous family of extracellular signaling molecules. It has been known for many years that adenosine diphosphate is a potent platelet aggregating factor, but it is now clear that virtually every circulating cell is responsive to nucleotides. Effects as different as proliferation or differentiation, chemotaxis, release ... More
The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells).
AuthorsArmand R, Channon JY, Kintner J, White KA, Miselis KA, Perez RP, Lewis LD
JournalToxicol Appl Pharmacol
PubMed ID15050409
'Mitochondrial DNA-deficient (rho(0)) cells were generated following a 26-day incubation of MOLT-4 lymphoblastoid T cells in ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide). The absence of mitochondrial DNA (mtDNA) in the resultant MOLT-4 rho(0) cells was confirmed by Southern analysis and quantitative polymerase chain reaction (PCR). MOLT-4 rho(0) cells proliferated more slowly than ... More
Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes.
AuthorsKapuscinski J
JournalJ Histochem Cytochem
PubMed ID1696951
'Interaction of cations with nucleic acids (NA) often results in condensation of the product. The driving force of aromatic cation-induced condensation is the cooperative interaction between ligand and single-stranded (ss) NA. This type of reaction is highly specific with regard to the primary and secondary structure of NA, and results ... More
Flow cytometry of DNA content and other indicators of proliferative activity.
AuthorsShapiro HM
JournalArch Pathol Lab Med
PubMed ID2471483
'Flow cytometric DNA content analysis, employing fluorescent stains that bind stoichiometrically to DNA, is widely used by researchers and clinicians to monitor cell growth kinetics and to detect abnormalities in nuclear DNA content in tumor cells. Measurement precision is critical because small differences in DNA content may be significant; plastic ... More
GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.
AuthorsMelikyan GB, White JM, Cohen FS
JournalJ Cell Biol
PubMed ID7593189
'Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). ... More
Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry.
AuthorsLatt SA
JournalJ Histochem Cytochem
PubMed ID70459
'Fluorometric detection of the biosynthetic incorporation of 5-bromodeoxyuridine (BrdU) into deoxyribonucleic acid has permitted cytologic studies of chromosome structure, replication, and repair. Some of these phenomena, previously detected using BrdU-dye techniques on fixed microscopic preparations, should be particularly amenable to analogous experimentation in fluorescence flow systems. Problems involved in interfacing ... More
Abundant alkali-sensitive sites in DNA of human and mouse sperm.
'The DNA of human and mouse sperm cells was analyzed by single-cell microgel electrophoresis, by agarose gel electrophoresis, and by alkaline elution--three techniques that can detect single-strand DNA breaks and/or labile sites. Under these conditions a surprisingly large number of single-strand DNA breaks, approximately 10(6) to 10(7) per genome, were ... More
Molecular flexibility of extended and compacted polynucleosomes. A steady-state fluorescence polarization study.
AuthorsHärd T, Nielsen PE, Norden B
JournalEur Biophys J
PubMed ID3234316
'We have studied the effects of Na+ (5-120 mM) and Mg2+ (0-6 mM) on the internal and overall flexibility of polynucleosome fragments from nuclease-solubilized chromatin from Ehrlich ascites cells. The mobility was monitored by the steady-state fluorescence polarization of the intercalated ethidium cation. The internal polynucleosome flexibility decreases continuously as ... More
Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells.
AuthorsBeisker W, Eisert WG
JournalJ Histochem Cytochem
PubMed ID2478614
'The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed ... More
Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants.
AuthorsBucana CD, Giavazzi R, Nayar R, O'Brian CA, Seid C, Earnest LE, Fan D
JournalExp Cell Res
PubMed ID2387329
'Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M). The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport. ... More
Petite induction in yeast, Saccharomyces cerevisiae, by phenanthridinium compounds: promotive effects of propidium iodide on mutagenesis by ethidium bromide or 8-deaminoethidium chloride.
AuthorsIwamoto Y, Yanagihara Y, Yielding LW, Yielding KL
JournalChem Pharm Bull (Tokyo)
PubMed ID3521920
Effects of ethidium bromide and bisbenzimide (Hoechst 33258) on human lymphocyte chromosome structure.
AuthorsAndersen O, Rønne M
JournalHereditas
PubMed ID2434451
Gene transfer from targeted liposomes to specific lymphoid cells by electroporation.
AuthorsMachy P, Lewis F, McMillan L, Jonak ZL
JournalProc Natl Acad Sci U S A
PubMed ID3186704
'Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell ... More
Capillary gel affinity electrophoresis of DNA fragments.
AuthorsGuttman A, Cooke N
JournalAnal Chem
PubMed ID1750704
'The incorporation of an affinity ligand within a polyacrylamide gel provides a general means of manipulating the selectivity of capillary gel electrophoresis separations. As an example of this approach, high resolution of DNA restriction fragments by capillary gel affinity electrophoresis has been achieved by adding a soluble intercalating agent, ethidium ... More
Replacement of odd bases in tRNA by fluorescent dyes.
AuthorsWintermeyer W, Zachau HG
JournalMethods Enzymol
PubMed ID4605047
A fluorescence probe of acetylcholine receptor conformation and local anesthetic binding.
AuthorsSchimerlik M, Raftery MA
JournalBiochem Biophys Res Commun
PubMed ID1008877
Measurement of electrophoretic mobility of dye-labeled large DNA fragments in agarose gels by movement of fluorescence pattern after photobleaching.
AuthorsChu B, Wang ZL, Wu C
JournalBiopolymers
PubMed ID2752103
Observation of individual DNA molecules undergoing gel electrophoresis.
AuthorsSmith SB, Aldridge PK, Callis JB
JournalScience
PubMed ID2911733
'Individual DNA molecules undergoing agarose gel electrophoresis were viewed with the aid of a fluorescence microscope. Molecular shape and orientation were studied in both steady and pulsed electric fields. It was observed that (i) DNA macromolecules advanced lengthwise through the gel in an extended configuration, (ii) the molecules alternately contracted ... More
Propidium iodide as a nuclear counterstain for immunofluorescence studies on cells in culture.
AuthorsJones KH, Kniss DA
JournalJ Histochem Cytochem
PubMed ID2432112
'We describe a rapid procedure using propidium iodide (PI) as a nuclear counterstain in immunofluorescence studies where cell surface or intracellular antigens are localized with fluorescein-conjugated antisera. In fixed monolayer preparations, all cell nuclei fluoresce red and can be seen simultaneously with cellular antigens that fluoresce green. Counterstaining with PI ... More
Inhibitory properties of nucleic acid-binding ligands on protein synthesis.
AuthorsMalina A, Khan S, Carlson CB, Svitkin Y, Harvey I, Sonenberg N, Beal PA, Pelletier J
JournalFEBS Lett
PubMed ID15620694
'The use of small molecule inhibitors in the study of cellular processes is a powerful approach to understanding gene function. During the course of a high throughput screen for novel inhibitors of eukaryotic translation, we identified a number of nucleic acid binding ligands that showed activity in our assay. When ... More
Terbium identifies double-stranded RNA on gels by quenching the fluorescence of intercalated ethidium bromide.
AuthorsAl-Hakeem M, Sommer SS
JournalAnal Biochem
PubMed ID3661991
'We report that the lanthanide cation terbium quenches the fluorescence of ethidium bromide bound to double-stranded RNA by 40-fold, whereas the quenching of double-stranded and single-stranded DNA is under 2.5-fold and the quenching of single-stranded RNA is under 5-fold. This observation was used to develop a convenient method of detecting ... More
Intercalation of ethidium bromide into a triple-stranded oligonucleotide.
AuthorsMergny JL, Collier D, Rougée M, Montenay-Garestier T, Hélène C
JournalNucleic Acids Res
PubMed ID2027760
'We have examined the ability of a cationic planar chromophore, ethidium bromide, to intercalate into a short, defined triple helix. Using UV absorption, fluorescence spectroscopy and a gel retardation assay we demonstrate that ethidium bromide is able to bind to a triple helix with a lower affinity than to the ... More
Theoretical study of ethidium intercalation in triple-stranded DNA and at triplex-duplex junctions.
'The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium- triple helix ... More
Latrunculin B or ATP depletion induces cofilin-dependent translocation of actin into nuclei of mast cells.
AuthorsPendleton A, Pope B, Weeds A, Koffer A
JournalJ Biol Chem
PubMed ID12566455
'Increasing cellular G-actin, using latrunculin B, in either intact or permeabilized rat peritoneal mast cells, caused translocation of both actin and an actin regulatory protein, cofilin, into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin ... More
The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans.
AuthorsGray DW, Morris PJ
JournalStain Technol
PubMed ID2448922
'There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets ... More
Cytofluorometric determination of protein-bound thiols and DNA in cell nuclei.
AuthorsSchabronath J, Streffer C, Zamboglou N
JournalCytometry
PubMed ID2340770
'The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein-bound sulfhydryl-groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N-iodoacetyl-N(5-sulfo-1-naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction ... More
A polarized photobleaching study of DNA reorientation in agarose gels.
AuthorsScalettar BA, Selvin PR, Axelrod D, Klein MP, Hearst JE
JournalBiochemistry
PubMed ID2364059
'Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions ... More
Review: ethidium fluorescence assay. Part II. Enzymatic studies and DNA-protein interactions.
AuthorsMorgan AR, Evans DH, Lee JS, Pulleyblank DE
JournalNucleic Acids Res
PubMed ID388344
'Almost all DNA and RNA metabolizing enzymes can be assayed rapidly and very sensitively by exploiting the enhanced fluorescence of ethidium intercalated into duplex DNA or RNA. Denatured DNA and natural RNAs contain duplex regions due to intramolecular hydrogen-bonding and can also be sensitively measured. Where the product is truly ... More
Fluorescent staining for leukocyte chemotaxis. Eosinophil-specific fluorescence with aniline blue.
AuthorsMcCrone EL, Lucey DR, Weller PF
JournalJ Immunol Methods
PubMed ID2460564
'To overcome problems associated with the quantitation of human eosinophil chemotaxis in micropore filters, we have developed a fluorescent method of specifically staining eosinophils in chemotactic filters. A neutral solution of aniline blue yielded bright green fluorescent staining of the cytoplasmic granules of eosinophils. Other leukocytes and contaminating neutrophils potentially ... More