Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry.
AuthorsHaaijman JJ, Coolen J, Kröse CJ, Pronk GJ, Ming ZF
JournalHistochemistry
PubMed ID3522496
'Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab''s was selected for immunohistology in which they functioned as a ... More
Prelysosomal acidic vacuoles in Dictyostelium discoideum.
AuthorsPadh H, Lavasa M, Steck TL
JournalJ Cell Biol
PubMed ID2466039
'We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. ... More
The multicollisional, obstructed, long-range diffusional nature of mitochondrial electron transport.
AuthorsChazotte B, Hackenbrock CR
JournalJ Biol Chem
PubMed ID3170548
'Data are presented which reveal that ubiquinone (Q)-mediated electron transport is a multicollisional, obstructed, long-range diffusion process, where factors that affect the rate of lateral diffusion also affect the rate of electron transport. Based on fluorescence recovery after photobleaching measurements, it was concluded that Q-mediated electron transport occurs by the ... More
Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.
AuthorsKnowles GC, McCulloch CA
JournalJ Histochem Cytochem
PubMed ID1527379
'Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ... More
Flow cytometric measurements of cytoplasmic calcium changes in human platelets.
AuthorsDavies TA, Drotts D, Weil GJ, Simons ER
JournalCytometry
PubMed ID3359892
'Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the ... More
Microinjection of fluorescently labeled alpha-actinin into living fibroblasts.
AuthorsFeramisco JR
JournalProc Natl Acad Sci U S A
PubMed ID291056
'alpha-Actinin from chicken gizzard labeled with tetramethylrhodamine isothiocyanate has been incorporated into living fibroblast cells by microinjection. Fluorescent labeling of alpha-actinin was carried out such that the conjugated protein was functional in vitro as shown by its ability to bind to F-actin. Within 1-2 hr after injection, diffuse fluorescence was ... More
Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin.
AuthorsHochman JH, Shimizu Y, DeMars R, Edidin M
JournalJ Immunol
PubMed ID2450918
'We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, ... More
Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization.
AuthorsKosk-Kosicka D, Bzdega T, Wawrzynow A
JournalJ Biol Chem
PubMed ID2531140
'Fluorescence resonance energy transfer has been used to study oligomerization of the purified erythrocyte Ca2+-ATPase. The energy transfer efficiency has been measured at different enzyme concentrations, from fluorescein 5''-isothiocyanate attached on one enzyme molecule to eosin 5-maleimide or tetramethylrhodamine 5-isothiocyanate attached on another enzyme molecule. The energy transfer efficiency showed ... More
Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes.
AuthorsDaukas G, Zigmond SH
JournalJ Cell Biol
PubMed ID4055891
'We have found that hypertonic medium inhibited the receptor-mediated uptake of the chemotactic peptide N-formylnorleucylleucylphenylalanine without affecting fluid-phase endocytosis by polymorphonuclear leukocytes (PMNs). Morphological and biochemical evidence demonstrated that cells in hypertonic medium did not accumulate peptide in a receptor-mediated manner. However, the cells continued to form endosomes containing fluid-phase ... More
The BCR/ABL oncogene alters the chemotactic response to stromal-derived factor-1alpha.
AuthorsSalgia R, Quackenbush E, Lin J, Souchkova N, Sattler M, Ewaniuk DS, Klucher KM, Daley GQ, Kraeft SK, Sackstein R, Alyea EP, von Andrian UH, Chen LB, Gutierrez-Ramos JC, Pendergast AM, Griffin JD
JournalBlood
PubMed ID10590068
'The chemokine stromal-derived factor-1alpha (SDF-1alpha) is a chemoattractant for CD34(+) progenitor cells, in vitro and in vivo. The receptor for SDF-1alpha, CXCR-4, is a 7 transmembrane domain receptor, which is also a coreceptor for human immunodeficiency virus (HIV). Here we show that transformation of hematopoietic cell lines by BCR/ABL significantly ... More
Effects of exposure to low pH on the lateral mobility of influenza hemagglutinin expressed at the cell surface: correlation between mobility inhibition and inactivation.
AuthorsGutman O, Danieli T, White JM, Henis YI
JournalBiochemistry
PubMed ID8418830
'To investigate the possible role of viral glycoprotein mobility in membrane fusion, fluorescence photobleaching recovery was employed to study the effects of exposure to mildly acidic pH (required to convert many viral fusion proteins to the fusion-active form) on the lateral mobility of influenza hemagglutinin (HA) proteins expressed at the ... More
Erratic deposition of agrin during the formation of Xenopus neuromuscular junctions in culture.
AuthorsAnderson MJ, Shi ZQ, Grawel R, Zackson SL
JournalDev Biol
PubMed ID7601300
'In order to disclose the mechanism that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses ... More
Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells.
AuthorsJenkins AL, Bootman MD, Taylor CW, Mackie EJ, Stone SR
JournalJ Biol Chem
PubMed ID8407986
'The receptor responsible for the increase in intracellular calcium concentration ([Ca2+]i) after the addition of thrombin to the human osteoblast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca2+]i; a half-maximal stimulation was observed with 3.2 +/- 1.1 nM thrombin. The human platelet thrombin receptor is ... More
Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp.
AuthorsHarman GE, Stasz TE
JournalStain Technol
PubMed ID2464211
'In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all ... More
Physical association between MHC class I and class II molecules detected on the cell surface by flow cytometric energy transfer.
AuthorsSzöllösi J, Damjanovich S, Balàzs M, Nagy P, Trón L, Fulwyler MJ, Brodsky FM
JournalJ Immunol
PubMed ID2732468
'The physical association of HLA class I and class II Ag in the membranes of PGF and JY lymphoblastoid cell lines was studied using flow cytometric energy transfer. This technique measures the proximity of cell surface molecules in the nm range and provides a distribution histogram of the average proximity ... More
Clustering, mobility, and triggering activity of small oligomers of immunoglobulin E on rat basophilic leukemia cells.
AuthorsMenon AK, Holowka D, Webb WW, Baird B
JournalJ Cell Biol
PubMed ID2418032
'We have recently shown that small oligomers of IgE bound to univalent receptors for IgE on the surface of rat basophilic leukemia cells induce extensive aggregation of the receptors at 4 degrees C into patches resolvable by fluorescence microscopy and that this does not occur with monomeric IgE (Menon, A. ... More
Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor.
AuthorsJohnson DA, Voet JG, Taylor P
JournalJ Biol Chem
PubMed ID6715368
'An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins ... More
Kinetic aspects of the aggregation of Clostridium perfringens theta-toxin on erythrocyte membranes. A fluorescence energy transfer study.
AuthorsHarris RW, Sims PJ, Tweten RK
JournalJ Biol Chem
PubMed ID2016307
'Fluorescence resonance energy transfer was used to monitor aggregation kinetics of the "thiol-activated" cytolysin (perfringolysin O (PFO) or theta-toxin) of Clostridium perfringens on erythrocyte membranes. PFO was labeled with the isothiocyanate derivatives of either fluorescein or tetramethylrhodamine. No detectable change in the hemolytic activity of PFO was detected after modification ... More
Microcontact printing of DNA molecules.
AuthorsLange SA, Benes V, Kern DP, Hörber JK, Bernard A
JournalAnal Chem
PubMed ID15018562
'The controlled placement of DNA molecules onto solid surfaces is the first step in the fabrication of DNA arrays. The sequential deposition of tiny drops containing the probe DNA fragments using arrays of spotting needles or ink jet nozzles has become a standard. However, a caveat of liquid spotting is ... More
Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.
AuthorsFishman MC, Dragsten PR, Spector I
JournalNature
PubMed ID6261153
'Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We ... More
Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.
'Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3''-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially ... More
Calmodulin stabilization of kinetochore microtubule structure to the effect of nocodazole.
AuthorsSweet SC, Rogers CM, Welsh MJ
JournalJ Cell Biol
PubMed ID3198685
'To investigate the function of calmodulin (CaM) in the mitotic apparatus, the effect of microinjected CaM and chemically modified CaMs on nocodazole-induced depolymerization of spindle microtubules was examined. When metaphase PtK1 cells were microinjected with CaM or a CaM-TRITC conjugate, kinetochore microtubules (kMTs) were protected from the effect of nocodazole. ... More
Evidence for allosteric linkage between exosites 1 and 2 of thrombin.
AuthorsFredenburgh JC, Stafford AR, Weitz JI
JournalJ Biol Chem
PubMed ID9325262
'Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and ... More
Endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen visualised using fluorescent conjugates.
AuthorsGarnett MC, Baldwin RW
JournalEur J Cell Biol
PubMed ID3093235
'The endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen has been investigated using several different fluorescent conjugates. These conjugates have been employed for both fluorescence microscopy to show the qualitative changes in distribution of antibody conjugates during endocytosis, and also flow cytofluorimetry to show the quantitative changes ... More
Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization.
AuthorsMenon AK, Holowka D, Webb WW, Baird B
JournalJ Cell Biol
PubMed ID2935543
'Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to ... More
C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states.
'Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments'' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the ... More
Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope.
AuthorsBrismar H, Trepte O, Ulfhake B
JournalJ Histochem Cytochem
PubMed ID7608524
'We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be ... More
Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues.
AuthorsGoodarzi K, Goodarzi M, Tager AM, Luster AD, von Andrian UH
JournalNat Immunol
PubMed ID12949533
'Leukotriene B4 (LTB4) is a potent chemoattractant for myeloid leukocytes, which express BLT1, the high-affinity receptor for LTB4. We report here that BLT1 is induced substantially in CD8+ effector T cells and at lower amounts in CD8+ central memory T cells. LTB4 elicited BLT1-dependent chemotaxis in effector cells, but not ... More
Two-step binding mechanism of fibrinogen to alpha IIb beta 3 integrin reconstituted into planar lipid bilayers.
AuthorsMüller B, Zerwes HG, Tangemann K, Peter J, Engel J
JournalJ Biol Chem
PubMed ID8454652
'The platelet integrin alpha IIb beta 3 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/1,2-dimyristoylphosphatidy lglycerol vesicles after removal of the detergent by adsorption ... More
Differential roles of Fc gamma RII and Fc gamma RIII in immune complex stimulation of human neutrophils.
AuthorsBrunkhorst BA, Strohmeier G, Lazzari K, Weil G, Melnick D, Fleit HB, Simons ER
JournalJ Biol Chem
PubMed ID1328206
'Insoluble immune complexes (IIC) stimulate human neutrophils through Fc gamma receptors. Freshly isolated human neutrophils express two FcR subclasses, FcRII and FcRIII. We explored the role of FcRII and FcRIII in this activation process by selectively binding each FcR subclass with the Fab fragments of the respective anti-FcR monoclonal antibodies ... More
Histochemical localization of estrogen and progesterone receptors: evaluation of a method.
AuthorsBergqvist A, Carlström K, Ljungberg O
JournalJ Histochem Cytochem
PubMed ID6715869
'A histochemical method for the detection of estrogen (ER) and progesterone (PR) receptors in human endometrium, using estrogen and progesterone derivatives linked to fluorochrome-labeled bovine serum albumin (E2-BSA-fluorescein isothiocyanate (FITC) and progesterone-BSA-tetramethylrhodamine isothiocyanate (TMRITC], has been evaluated. The fluorochrome-labeled steroids were bound to the cytoplasm--preferably in glandular epithelial cells but ... More
Major histocompatibility complex class I protein conformation altered by transmembrane potential changes.
AuthorsBene L, Szöllósi J, Balázs M, Mátyus L, Gáspár R, Ameloot M, Dale RE, Damjanovich S
JournalCytometry
PubMed ID9098627
'The nature of charge distributions in membrane-bound macromolecular structures renders them susceptible to interaction with transmembrane potential fields. As a result, conformational changes in such species may be expected to occur when this potential is altered. We have detected reversible conformational change in the major histocompatibility complex (MHC) class I ... More
Membrane changes in neural target cells studied with fluorescent lectin probes.
AuthorsGualandris L, Rougé P, Duprat AM
JournalJ Embryol Exp Morphol
PubMed ID6655431
'The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic differences in their morphology, their composition and the molecular arrangement of the various constituents. The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v. microscopy (epi-illumination) and the quantification of the results show ... More
Simultaneous use of contrasting fluorochromes to separate measles and canine distemper viruses in a common system.
AuthorsGourlay JA, Pemberton JR
JournalAppl Microbiol
PubMed ID5000868
'Measles and canine distemper viruses were grown together in a Vero monkey kidney cell line. Each virus could be identified and individually titrated by using the color contrast produced by the reddish tetra-methyl rhodamine isothiocyanate-tagged antimeasles conjugate and the green fluorescein isothiocyanate-tagged antidistemper conjugate. Both blue light and green light ... More
Use of fluorescent antibodies in the study of lymphoid cell membrane molecules.
AuthorsForni L, de Petris S
JournalMethods Enzymol
PubMed ID6084798
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Photobleaching recovery studies of membrane events accompanying lectin stimulation of rabbit lymphocytes.
AuthorsLeuther MD, Barisas BG, Peacock JS, Krakauer H
JournalBiochem Biophys Res Commun
PubMed ID314290
The synthesis and use of fluorescent oligonucleotides in DNA sequence analysis.
AuthorsSmith LM, Kaiser RJ, Sanders JZ, Hood LE
JournalMethods Enzymol
PubMed ID3431463
Three-dimensional imaging in fluorescence by confocal scanning microscopy.
AuthorsBrakenhoff GJ, van der Voort HT, van Spronsen EA, Nanninga N
JournalJ Microsc
PubMed ID2651673
'The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.' ... More
Novel tools for the study of development, migration and turnover of nematocytes (cnidarian stinging cells).
AuthorsWeber J
JournalJ Cell Sci
PubMed ID7738115
'The rhodamine derivatives tetramethyl-rhodamine-5/6-maleimide (TROMI) and tetramethyl-rhodamine-6-isothiocyanate (TRITC) were allowed to react with living Hydra vulgaris. The two fluorescent dyes stain the polyps to different degrees, apparently without impairing their viability and behaviour. Concerning nematocytes, TROMI preferentially couples to cytoskeletal elements only of mounted nematocytes whereas TRITC selectively reacts with ... More
Connective tissue remodeling in corneal and scleral wounds.
AuthorsDavison PF, Galbavy EJ
JournalInvest Ophthalmol Vis Sci
PubMed ID3759366
'The fluorescent dye dichlorotriazinyl aminofluorescein will bind to amino groups of proteins covalently under physiological conditions. It has been used to dye the connective tissue around an ulcer or nonpenetrating, linear incision in the rabbit cornea and sclera, and the healing of the tissue has been examined up to 1 ... More
Simultaneous measurement of spectroscopic and physiological signals from a planar bilayer system: detecting voltage-dependent movement of a membrane-incorporated peptide.
AuthorsHanyu Y, Yamada T, Matsumoto G
JournalBiochemistry
PubMed ID9799498
'We developed an experimental system that can measure spectroscopic and physiological signals simultaneously from ion channels in a planar lipid bilayer, to study the relationship between the structure and function of the ion channels. While the membrane potential was clamped, fluorescent emission and ionic currents were measured simultaneously. The fluorescent ... More
Neuronal domains in developing neocortex.
AuthorsYuste R, Peinado A, Katz LC
JournalScience
PubMed ID1496379
'The mammalian neocortex consists of a mosaic of columnar units whose development is poorly understood. Optical recordings of brain slices labeled with the fluorescent calcium indicator fura-2 revealed that the neonatal rat cortex was partitioned into distinct domains of spontaneously coactive neurons. In tangential slices, these domains were 50 to ... More
An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin.
AuthorsWarn RM, Flegg L, Warn A
JournalJ Cell Biol
PubMed ID3117804
'Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At ... More
Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization.
AuthorsOkamura Y, Kondo S, Sase I, Suga T, Mise K, Furusawa I, Kawakami S, Watanabe Y
JournalNucleic Acids Res
PubMed ID11121494
'A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to ... More
Synthetic peptide GRGDS induces dissociation of alpha-actinin and vinculin from the sites of focal contacts.
AuthorsStickel SK, Wang YL
JournalJ Cell Biol
PubMed ID3138248
'The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were ... More
Binding of IgG to MoFc gamma RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy.
AuthorsPoglitsch CL, Sumner MT, Thompson NL
JournalBiochemistry
PubMed ID1829634
'Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the ... More
Biotin and fluorescent labeling of RNA using T4 RNA ligase.
AuthorsRichardson RW, Gumport RI
JournalNucleic Acids Res
PubMed ID6194506
'Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5''-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3''-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. ... More
Analysis of interaction between chaperonin GroEL and its substrate using fluorescence correlation spectroscopy.
AuthorsPack CG, Nishimura G, Tamura M, Aoki K, Taguchi H, Yoshida M, Kinjo M
JournalCytometry
PubMed ID10404975
'Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion properties of fluorescent molecules in tiny detection volume and allows the analysis of binding processes of biomolecules in homogeneous solution. In this study, FCS was used to measure equilibrium binding constants of disulfide-reduced apo-alpha-lactalbumin (rLA), denatured pepsin, and apo-cytochrome c (apo-cyt ... More
Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal.
AuthorsWen W, Harootunian AT, Adams SR, Feramisco J, Tsien RY, Meinkoth JL, Taylor SS
JournalJ Biol Chem
PubMed ID7798221
'The heat-stable inhibitor of cAMP-dependent protein kinase (PKI) was shown previously to export the kinase catalytic subunit (C) from the nucleus (Fantozzi, D. A., Harootunian, A. T., Wen, W., Taylor, S. S., Feramisco, J.R., Tsien, R. Y., and Meinkoth, J. L. (1994) J. Biol. Chem. 269, 2676-2686), in addition to ... More
Chemical methods of DNA and RNA fluorescent labeling.
AuthorsProudnikov D, Mirzabekov A
JournalNucleic Acids Res
PubMed ID8948646
'Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3''-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde ... More
Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.
AuthorsTonkinson JL, Stein CA
JournalNucleic Acids Res
PubMed ID7937155
'We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was ... More
Early membrane rupture events during neutrophil-mediated antibody-dependent tumor cell cytolysis.
AuthorsKindzelskii AL, Petty HR
JournalJ Immunol
PubMed ID10092769
'Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells ... More
Conjugation of antibodies with fluorochromes: modifications to the standard methods.
AuthorsGoding JW
JournalJ Immunol Methods
PubMed ID796385
'A number of modification to the standard procedures for coupling of fluorochromes to antibodies are described. The suggested procedures result in economies of time, labour and materials, and allow the reliable production of high quality conjugates. The modifications include the use of staphylococcal protein A-Sepharose for (a) a simple one-step ... More
Fluorescent dyes demonstrate the uniform expansion of the growing rabbit cornea.
AuthorsDavison PF, Galbavy EJ
JournalInvest Ophthalmol Vis Sci
PubMed ID4030248
'Thiocyanate and triazinyl chloride derivatives of fluorescent dyes have been employed for the covalent labeling of components of the connective tissue of the rabbit cornea. Collagen is the major macromolecular component that becomes dyed. Some of the stromal components that are labeled by these reagents in the first two weeks ... More
The absence of specific dye-coupling among frog spinal neurons.
AuthorsPowell SL, Westerfield M
JournalBrain Res
PubMed ID6607766
'A double fluorescence labeling technique was developed to study the specificity of dye-coupling among frog spinal neurons. A pool of motoneurons known to be electrically coupled was prelabeled with a large molecule (rhodamine conjugated to horseradish peroxidase) that was not expected to pass through gap junctions. Then a single sensory ... More
Fluorescence detection in automated DNA sequence analysis.
AuthorsSmith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE
JournalNature
PubMed ID3713851
'We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific ... More
Interactions of molecular probes with living cells and tissues. Part 1. Some general mechanistic proposals, making use of a simplistic Chinese box model.
AuthorsHorobin RW, Rashid F
JournalHistochemistry
PubMed ID2358379
'A simple and generalised model-termed the simplistic Chinese box [SCB] model-for the interaction of molecular probes with living systems is described. The SCB model includes the following assumptions. That living systems may be considered as built from biologically defined boxes, e.g. whole cell, nucleus, nucleoli. That movement of molecular probes ... More
Hydrophobic adsorption chromatography to reduce nonspecific staining by rhodamine-labeled antibodies.
AuthorsSpack EG, Packard B, Wier ML, Edidin M
JournalAnal Biochem
PubMed ID2432803
'Rhodamine absorbed to protein was removed from rhodamine-conjugated antibody solutions by adsorption to hydrophobic macroporous beads (Bio-Beads SM-2) following gel permeation chromatography. This simple technique eliminated the contaminating free dye more effectively than gel filtration alone, but neither significantly reduced the level of fluorescently conjugated antibody nor altered the latter''s ... More
The use of fluorescent probes in immunochemistry.
AuthorsDavidson RS, Hilchenbach MM
JournalPhotochem Photobiol
PubMed ID2217553
'The limitations and advantages of particular dyes for labelling proteins and other biological materials are discussed. Methods available for conjugating dyes to proteins are outlined. Following a discussion of double labelling methods the use of photoactivatable fluorochromes and time resolved fluorescence methodologies are outlined. The reasons for the photoinstability of ... More
Single-molecule tracking of mRNA exiting from RNA polymerase II.
AuthorsAndrecka J, Lewis R, Brückner F, Lehmann E, Cramer P, Michaelis J,
JournalProc Natl Acad Sci U S A
PubMed ID18162559
'Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5'' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active ... More
Three-color immunofluorescence histochemistry allowing triple labeling within a single section.
AuthorsStaines WA, Meister B, Melander T, Nagy JI, Hökfelt T
JournalJ Histochem Cytochem
PubMed ID2891745
'We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances ... More
CR3 (alphaM beta2; CD11b/CD18) restores IgG-dependent phagocytosis in transfectants expressing a phagocytosis-defective Fc gammaRIIA (CD32) tail-minus mutant.
AuthorsWorth RG, Mayo-Bond L, van de Winkel JG, Todd RF, Petty HR
JournalJ Immunol
PubMed ID8955219
'Previous studies have suggested that complement receptors cooperate with Fc receptors to mediate Ab-dependent effector functions. In the present study, we tested the capacity of complement receptor type 3 (CR3; alphaM beta2; CD11b/CD18) to participate in Fc gammaRIIA (CD32)-mediated phagocytosis. To test this hypothesis, we transfected a phagocytosis-defective tail-minus mutant ... More
Cycling of the integral membrane glycoprotein, LEP100, between plasma membrane and lysosomes: kinetic and morphological analysis.
AuthorsLippincott-Schwartz J, Fambrough DM
JournalCell
PubMed ID3107839
'LEP100 (an integral membrane glycoprotein, Mr = 100,000) occurs in three subcellular compartments: lysosome (approximately 90% of the molecules), endosome (5%-8%), and plasma membrane (2%-3%). Rate constants for movement to and from each compartment have been estimated. The movement of LEP100 from endosomes to lysosomes was blocked by chloroquine, causing ... More
Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus.
AuthorsFantozzi DA, Harootunian AT, Wen W, Taylor SS, Feramisco JR, Tsien RY, Meinkoth JL
JournalJ Biol Chem
PubMed ID8300597
'The catalytic (C) subunit of cAMP-dependent protein kinase is inhibited by the regulatory (R) subunit and by a thermostable inhibitor (PKI). Both inhibitors also affect the intracellular distribution of the C subunit. Whether injected into the cytoplasm or into the nucleus, free C subunit can enter and exit the nucleus ... More
Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.
'The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different ... More
Simultaneous demonstration of catecholamines and phenylethanolamine-N-methyltransferase in the same tissue section by means of formaldehyde-induced fluorescence (FIF) and tetramethyl-rhodamine-isothiocyanate (MRITC) immunofluorescence.
AuthorsSchröder H, Lackner K, Heym C
JournalJ Neurosci Methods
PubMed ID6755083
Calmodulin purification and fluorescent labeling.
AuthorsDedman JR, Kaetzel MA
JournalMethods Enzymol
PubMed ID6645966
The conjugation of immunoglobulins with tetramethylrhodamine isothiocyanate. A comparison between the amorphous and the crystalline fluorochrome.
AuthorsAmante L, Ancona A, Forni L
JournalJ Immunol Methods
PubMed ID4561342
Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization.
AuthorsRavdin P, Axelrod D
JournalAnal Biochem
PubMed ID889094
Biologically active derivatives of angiotensin for labeling cellular receptors.
AuthorsGalardy RE, Stafford SS, Schaefer ML, Ho H, La Vorgna KA, Jamieson JD
JournalJ Med Chem
PubMed ID214560
Membrane receptor mobility changes by Sendai virus.
Determination of the molar absorbance coefficient of bound tetramethyl rhodamine isothiocyanate relative to fluorescein isothiocyanate.
Authorsvan Dalen JP, Haaijman JJ
JournalJ Immunol Methods
PubMed ID4601411
Distribution of fluorescently labeled alpha-actinin in living and fixed fibroblasts.
AuthorsFeramisco JR, Blose SH
JournalJ Cell Biol
PubMed ID7190570
Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.
AuthorsBrelje TC, Wessendorf MW, Sorenson RL
JournalMethods Cell Biol
PubMed ID8246789
Preparation of a fluorescent analog: acetamidofluoresceinyl-labeled Dictyostelium discoideum alpha-actinin.
AuthorsSimon JR, Taylor DL
JournalMethods Enzymol
PubMed ID3029546
Fluorescence microphotolysis. Diffusion measurements in single cells.
AuthorsPeters R
JournalNaturwissenschaften
PubMed ID6877390
Homogeneous immunochemical technique for determination of human lactoferrin using excitation transfer and phase-resolved fluorometry.
AuthorsNithipatikom K, McGown LB
JournalAnal Chem
PubMed ID3551686
The use of microinjection and video microscopy for the study of calmodulin and calcium in living cells.
AuthorsShelanski M, Ratan R
JournalMethods Enzymol
PubMed ID3587048
Detection of doubly stained fluorescent specimens using confocal microscopy.
AuthorsMossberg K, Ericsson M
JournalJ Microsc
PubMed ID1695246
Studies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and ... More
Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides.
AuthorsAgrawal S, Christodoulou C, Gait MJ
JournalNucleic Acids Res
PubMed ID3748808
The syntheses are described of two types of linker molecule useful for the specific attachment of non-radioactive labels such as biotin and fluorophores to the 5' terminus of synthetic oligodeoxyribonucleotides. The linkers are designed such that they can be coupled to the oligonucleotide as a final step in solid-phase synthesis ... More
Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts.
AuthorsBarsby TL, Yarrow SA, Shepard JF
JournalStain Technol
PubMed ID6495350
Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous ... More
Computerized analysis of TV images for ultrasensitive monitoring of the reaction of fluorochrome with protein.
AuthorsRees DD, Fogarty KE, Levy LK, Fay FS
JournalAnal Biochem
PubMed ID3993911
Preparation of protein-specific fluorescent probes with the desired degree of fluorochrome can be greatly facilitated by a technique that combines thin-layer chromatography (TLC) with quantitative image analysis (QIA). Using TLC/QIA, the investigator can determine the fluorochrome/protein ratio on-line with only a few micrograms of protein as fluorochrome is conjugated to ... More
Alternative fluorochromes to ethidium bromide for automated read out of cytotoxicity tests.
AuthorsTanke HJ, van der Linden PW, Langerak J
JournalJ Immunol Methods
PubMed ID6749996
Propidium iodide (PI) and diamidinophenylindol (DAPI) were compared with ethidium bromide (EB) in cytotoxicity tests. Double blind studies showed similar results with the 3 dyes in terms of percentages of dead (fluorescing) cells. The dyes were also investigated for their potential combination with the immunofluorescent membrane markers FITC and TRITC. ... More
Stabilization of fluorochromes after immunofluorescent staining of tissue and embedding in Epon.
AuthorsEneström S
JournalStain Technol
PubMed ID3087035
Tissue blocks or sections immunofluorescent stained before embedding, i.g., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so ... More
Synaptotagmin regulation of coated pit assembly.
Authorsvon Poser C, Zhang JZ, Mineo C, Ding W, Ying Y, Sudhof TC, Anderson RG
JournalJ Biol Chem
PubMed ID10906143
Synaptotagmins bind clathrin AP-2 with high affinity via their second C(2) domain, which indicates they are involved in coated pit function. We now report that expression of synaptotagmins lacking either the second C(2) domain or the entire cytoplasmic region potently inhibit endocytosis. Inhibition was dependent on two intramembrane cysteine residues ... More
Two-dimensional electrochromatography/capillary electrophoresis on a microchip.
AuthorsGottschlich N, Jacobson SC, Culbertson CT, Ramsey JM
JournalAnal Chem
PubMed ID11403315
A two-dimensional separation system on a microfabricated device was demonstrated using open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension was operated under isocratic conditions, and the effluent from the first dimension was repetitively injected into the second dimension every few seconds. A ... More
The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.
AuthorsSmith LM, Fung S, Hunkapiller MW, Hunkapiller TJ, Hood LE
JournalNucleic Acids Res
PubMed ID4000959
A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to ... More
Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.
AuthorsBraun-Howland EB, Danielsen SA, Nierzwicki-Bauer SA
JournalBiotechniques
PubMed ID1282348
A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests ... More
An approach to quantitation in rhodamine isothiocyanate labeling.
AuthorsMcKinney RM, Spillane JT
JournalAnn N Y Acad Sci
PubMed ID52324
A survey of commercially available TMRI and RBI products revealed that many are of unsatisfactory quality. Optimum dye/IgG ratios were obtained in conjugates with TMRI of about 65% purity by labeling them with reaction mixture ratios of 10 and 20 mug dye/mg IgG. Optimum labeling with RBI of comparable purity ... More
Double staining technique using a combination of indirect and direct immunofluorescence with monoclonal antibodies.
AuthorsKupper H, Storz H
JournalActa Histochem
PubMed ID2425537
A simple method for double staining by immunofluorescence is described. If for double staining using monoclonal antibodies of the same species only one antibody is conjugated with FITC or TRITC, a combination of indirect and direct immunofluorescence is possible. For cell staining the following incubation steps are carried out: Monoclonal ... More
The distribution of calmodulin in living mitotic cells.
AuthorsZavortink M, Welsh MJ, McIntosh JR
JournalExp Cell Res
PubMed ID6641808
Calmodulin has been labeled with rhodamine isothiocyanate (CaM-RITC) and used as a probe for the location of calmodulin in vivo. CaM-RITC retains its capacity to regulate the activity of brain phosphodiesterase in a Ca2+-dependent manner in vitro, indicating that the labeled protein is still active. After injection into living mammalian ... More
Photoaffinity labeling of antibodies for applications in homogeneous fluoroimmunoassays.
AuthorsChang IN, Lin JN, Andrade JD, Herron JN
JournalAnal Chem
PubMed ID7762830
A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and ... More
Rhodamine conjugates:specific and nonspecific binding properties in immunohistochemistry.
AuthorsBrandtzaeg P
JournalAnn N Y Acad Sci
PubMed ID52316
Anionic-exchange fractions of IgG labeled with FITC, MRITC, RB200SC, or RBITC were tested on different substrates, and the resultant fluorescence was evaluated with the Ploem optical system. Conjugations with MRITC or RB200SC were found to afford the following advantages over FITC: immunofluorescence sensitivity was elevated six to seven times on ... More
Dynamic isoelectric focusing for proteomics.
AuthorsMontgomery R, Jia X, Tolley L
JournalAnal Chem
PubMed ID16970328
Dynamic isoelectric focusing is a new technique that is related to capillary isoelectric focusing but uses additional high-voltage power supplies to provide control over the shape of the electric field within the capillary. Manipulation of the electric field changes the pH gradient, enabling both the location and width of the ... More
Yeast proteins that recognize nuclear localization sequences.
AuthorsSilver P, Sadler I, Osborne MA
JournalJ Cell Biol
PubMed ID2670959
A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Ga14 and histone H2B. These proteins are assayed by the binding ... More
Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts.
AuthorsEldridge CA, Elson EL, Webb WW
JournalBiochemistry
PubMed ID6246920
Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in ... More
Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.
AuthorsWessendorf MW, Brelje TC
JournalHistochemistry
PubMed ID1429023
There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin ... More
Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP.
AuthorsSabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, Balbin M, Lopez-Otin C, Shapiro S, Inada M, Krane S, Allen E, Chung D, Weiss SJ
JournalJ Cell Biol
PubMed ID15557125
As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from ... More
Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies.
AuthorsWessel GM, McClay DR
JournalJ Histochem Cytochem
PubMed ID3084626
We identify two tissue-specific molecules in the sea urchin embryo by an immunofluorescence technique capable of co-localizing monoclonal antibodies on the same tissue section. The technique uses monovalent Fab-fluorochrome conjugates as secondary reagents to avoid cross-talk of subsequent antibody probes. Using this technique, we show that two cell surface molecules ... More