Detyrosination of alpha tubulin does not stabilize microtubules in vivo.
AuthorsWebster DR, Wehland J, Weber K, Borisy GG
JournalJ Cell Biol
PubMed ID1973168
'The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h ... More
Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.
'A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading ... More
Steady-state fluorescence studies on lipase-vesicle interactions.
AuthorsMosmuller EW, Pap EH, Visser AJ, Engbersen JF
JournalBiochim Biophys Acta
PubMed ID8305458
'The interaction of lipase from Candida cylindracea (CCL) with positively charged polymerizable surfactant vesicles was studied by the use of steady-state fluorescence techniques. The phase transition of vesicles composed of nonpolymerized and polymerized N-allylbis[2-(hexadecanoyloxy)ethyl]methylammonium bromide (ABHEMA Br) was determined in the absence of lipase, by measuring the change in fluorescence ... More
A test of microtubule translocation during neurite elongation.
AuthorsLim SS, Edson KJ, Letourneau PC, Borisy GG
JournalJ Cell Biol
PubMed ID2195037
'In a previous study using PC-12 cells (Lim, S. S., P. J. Sammak, and G. G. Borisy, 1989. J. Cell Biol. 109:253-263), we presented evidence that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary. However, neurites of PC-12 cells grow slowly, hindering a stringent test of ... More
Fluorescent differential display: arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.
AuthorsIto T, Kito K, Adati N, Mitsui Y, Hagiwara H, Sakaki Y
JournalFEBS Lett
PubMed ID7521850
'We established robust, reliable protocols for ''Differential Display (DD),'' an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967-971] using RT-PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as ... More
Fluorescent localization of contractile proteins in tissue culture cells.
AuthorsWang K, Feramisco JR, Ash JF
JournalMethods Enzymol
PubMed ID6750319
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
Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies.
AuthorsTitus JA, Haugland R, Sharrow SO, Segal DM
JournalJ Immunol Methods
PubMed ID6806389
The sulfonylchloride derivative of the red-emitting fluorophore, sulforhodamine 101, has been synthesized in order to provide a reagent for coupling to amino groups on proteins and other compounds, and it is now commercially available under the name 'Texas Red'. Texas Red conjugates of antibodies and other proteins have been prepared ... More
A dual laser analysis of the migration of XRITC-labeled, FITC-labeled, and double-labeled lymphocytes in sheep.
AuthorsAbernethy NJ, Chin W, Lyons H, Hay JB
JournalCytometry
PubMed ID3930188
Substituted rhodamine isothiocyanate (XRITC) has been used to study lymphocyte migration in sheep. After being labeled in vitro with XRITC, lymphocytes appeared in the efferent lymph of single lymph nodes with the same kinetics as cells labeled with fluorescein isothiocyanate (FITC). The recovery of intravenously injected XRITC-labeled cells was followed ... More
The position of the ATP binding site on the (Ca2+ + Mg2+)-ATPase.
AuthorsGutierrez-Merino C, Munkonge F, Mata AM, East JM, Levinson BL, Napier RM, Lee AG
JournalBiochim Biophys Acta
PubMed ID2949777
We present a convenient method to calculate the efficiency of fluorescence energy transfer in two-dimensional membrane systems. We apply it to the analysis of energy transfer between phospholipid molecules labelled with fluorescein and rhodamine groups, and of energy transfer in reconstituted membranes containing (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, ... More
Direct measurement of microvessel hematocrit, red cell flux, velocity, and transit time.
AuthorsSarelius IH, Duling BR
JournalAm J Physiol
PubMed ID7149038
A method is presented for the in vivo study of red cell flow dynamics. The method permits direct measurement of the red cell volume fraction in microvessel blood without resort to in vitro calibration curves. Furthermore, the method does not require extensive mathematical manipulation and can be applied to any ... More
The measurement of specific cell: cell interactions by dual-parameter flow cytometry.
AuthorsSegal DM, Stephany DA
JournalCytometry
PubMed ID6370631
The Fc receptor-mediated aggregation of antibody-coated spleen cells with cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green-emitting (fluorescein isothiocyanate) and red-emitting (substituted rhodamine isothiocyanate) fluorophores, respectively. They were mixed, incubated in suspension at 4 ... More
Photodamage to intact erythrocyte membranes at high laser intensities: methods of assay and suppression.
AuthorsBloom JA, Webb WW
JournalJ Histochem Cytochem
PubMed ID6725935
A simple hemolytic assay of the photodamage suffered by individual intact erythrocytes upon localized exposure to high laser intensities such as those encountered in fluorescence photobleaching recovery (FPR) experiments has been characterized. At incident beam powers over 100,000 W/cm2 at 514 or 568 nm, hemoglobin absorption induces thermal-shock lysis. Below ... More
Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides.
Phosphorothioate oligodeoxynucleotides (P=S ODNs) are frequently used as antisense agents to specifically interfere with the expression of cellular target genes. However, the cell biological properties of P=S ODNs are poorly understood. Here we show that P=S ODNs were able to continuously shuttle between the nucleus and the cytoplasm and that ... More
Accessory factors facilitate the binding of glucocorticoid receptor to the phosphoenolpyruvate carboxykinase gene promoter.
Glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene requires a glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three accessory factor elements (gAF1-3). DNA-binding accessory proteins are commonly required for the regulation of genes whose products play an important ... More
Rapid, one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry.
AuthorsCrissman HA, Steinkamp JA
JournalCytometry
PubMed ID6216083
Detailed, simplified techniques are described for simultaneous staining and analysis of DNA and protein in a number of mammalian cell types. Cell staining involves the addition of appropriate dye mixtures to unfixed or ethanol-fixed cells and subsequent analysis of cell populations in the staining reagents generally within 10 to 20 ... More
A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp.
AuthorsAnderson SG, Williams CR, O'donnell M, Bloom LB
JournalJ Biol Chem
PubMed ID17210572
Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by ... More
A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader.
AuthorsAson B, Bertram JG, Hingorani MM, Beechem JM, O'Donnell M, Goodman MF, Bloom LB
JournalJ Biol Chem
PubMed ID10644772
The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. ... More
A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification.
AuthorsCain CC, Murphy RF
JournalJ Cell Biol
PubMed ID2892844
To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic ... More
Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers.
Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids. We describe a carrier system for intracellular ... More
The mechanism of intercellular aggregation. I. The kinetics of the Fc gamma receptor-mediated aggregation of P388D1 cells with antibody-coated lymphocytes at 4 degrees C.
AuthorsSegal DM, Stephany DA
JournalJ Immunol
PubMed ID6230397
The formation of specific, heterophilic conjugates between cells from the P388D1 mouse macrophage line and antibody-coated mouse spleen cells was followed in cell suspensions at 4 degrees C by dual parameter flow cytometry. Intercellular aggregation in this system is mediated by the binding of the Fc portions of IgG antibodies ... More
Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase.
AuthorsOtto MR, Bloom LB, Goodman MF, Beechem JM
JournalBiochemistry
PubMed ID9665721
DNA polymerases are complex enzymes which bind primer-template DNA and subsequently either extend or excise the terminal nucleotide on the primer strand. In this study, a stopped-flow fluorescence anisotropy binding assay is combined with real-time measurements of a fluorescent adenine analogue (2-aminopurine) located at the 3'-primer terminus. Using this combined ... More
Measurement of the absolute temporal coupling between DNA binding and base flipping.
AuthorsAllan BW, Reich NO, Beechem JM
JournalBiochemistry
PubMed ID10220317
The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1 </= kon </= 2.25 x 10(7) M-1 s-1. Base-flipping kinetics (monitored using 2-aminopurine ... More
Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics.
AuthorsPerez-Howard GM, Weil PA, Beechem JM
JournalBiochemistry
PubMed ID7794913
A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation ... More
Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations.
AuthorsAhlers M, Grainger DW, Herron JN, Lim K, Ringsdorf H, Salesse C
JournalBiophys J
PubMed ID1420916
Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4-4-20 generated against the hapten, fluorescein, in these membrane models ... More
Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.
AuthorsSubirade M, Salesse C, Marion D, Pézolet M
JournalBiophys J
PubMed ID8519997
The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the ... More