Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface.
AuthorsDíaz JF, Barasoain I, Souto AA, Amat-Guerri F, Andreu JM
JournalJ Biol Chem
PubMed ID15550392
'The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4''-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed ... More
High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif.
AuthorsLi SC, Songyang Z, Vincent SJ, Zwahlen C, Wiley S, Cantley L, Kay LE, Forman-Kay J, Pawson T
JournalProc Natl Acad Sci U S A
PubMed ID9207069
'The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination ... More
The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts.
AuthorsBurgess TL, Qian Y, Kaufman S, Ring BD, Van G, Capparelli C, Kelley M, Hsu H, Boyle WJ, Dunstan CR, Hu S, Lacey DL
JournalJ Cell Biol
PubMed ID10225954
'Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. ... More
Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores.
AuthorsRozinov MN, Nolan GP
JournalChem Biol
PubMed ID9862799
'BACKGROUND: Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo. RESULTS: A panel of Texas red, Rhodamine red, Oregon green ... More
Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance.
AuthorsFidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE
JournalMol Cell
PubMed ID11090624
'The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, ... More
General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis.
AuthorsCraig DB, Wong JC, Polakowski R, Dovichi NJ
JournalAnal Chem
PubMed ID9751024
'Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate ... More
Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics.
AuthorsTiyanont K, Doan T, Lazarus MB, Fang X, Rudner DZ, Walker S
JournalProc Natl Acad Sci U S A
PubMed ID16832063
'The peptidoglycan (PG) layers surrounding bacterial cells play an important role in determining cell shape. The machinery controlling when and where new PG is made is not understood, but is proposed to involve interactions between bacterial actin homologs such as Mbl, which forms helical cables within cells, and extracellular multiprotein ... More
Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery.
'A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic ... More
Evidence for an upper affinity threshold for anti-IgM-induced apoptosis in a human B-cell lymphoma.
AuthorsMongini PK, Liu Q, Vilensky MA, Highet PF, Inman JK
JournalBlood
PubMed ID9808570
'The influence of ligand:receptor affinity on B-cell antigen receptor (BCR)-induced apoptosis in the IgM+ Burkitt lymphoma line, Ramos, was evaluated with a group of affinity-diverse murine monoclonal antibodies (MoAbs) specific for human B-cell IgM. The studies showed not only a minimal affinity threshold for the induction of apoptosis, but, interestingly, ... More
Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction.
AuthorsDornan S, Sebestyen Z, Gamble J, Nagy P, Bodnar A, Alldridge L, Doe S, Holmes N, Goff LK, Beverley P, Szollosi J, Alexander DR
JournalJ Biol Chem
PubMed ID11694532
'An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was ... More
Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection.
AuthorsCruickshank KA
JournalAnal Biochem
PubMed ID10094771
'A method has been developed by which enzymatically incorporated fluorophore-labeled nucleotide sites in nucleic acid can be quantitated by degradation of nanogram quantities of DNA followed by capillary gel electrophoretic analysis with fluorescence detection. In this way the differing relative labeling densities achieved using either C5-substituted dUTP''s or N4-substituted dCTP''s ... More
Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.
AuthorsCraig DB, Polakowski RM, Arriaga E, Wong JC, Ahmadzadeh H, Stathakis C, Dovichi NJ
JournalElectrophoresis
PubMed ID9761200
We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample ... More
Antibody-antigen binding constants determined in solution-phase with the threshold membrane-capture system: binding constants for anti-fluorescein, anti-saxitoxin, and anti-ricin antibodies.
AuthorsDill K, Lin M, Poteras C, Fraser C, Hafeman DG, Owicki JC, Olson JD
JournalAnal Biochem
PubMed ID8203727
Affinities of various monoclonal and polyclonal antibodies for fluorescein-containing antigens, saxitoxin and ricin, were determined by using a light addressable potentiometric sensor-based system (Threshold). The dissociation constants, determined from Scatchard plots, ranged from 2 x 10(-7) to approximately 3 x 10(-12) M. Dissociation constants for fluorescein and saxitoxin were compared ... More
Squid hnRNP protein promotes apical cytoplasmic transport and localization of Drosophila pair-rule transcripts.
AuthorsLall S, Francis-Lang H, Flament A, Norvell A, Schüpbach T, Ish-Horowicz D
JournalCell
PubMed ID10428029
Drosophila melanogaster pair-rule segmentation gene transcripts localize apically of nuclei in blastoderm embryos. This might occur by asymmetric (vectorial) export from one side of the nucleus or by transport within the cytoplasm. We have followed fluorescently labeled pair-rule transcripts postinjection into Drosophila embryos. Naked, microinjected fushi tarazu (ftz) transcripts do ... More
9-Anthroylnitrile binding to serine-181 in myosin subfragment 1 as revealed by FRET spectroscopy and molecular modeling.
AuthorsSzarka K, Bódis E, Visegrády B, Nyitrai M, Kilár F, Somogyi B
JournalBiochemistry
PubMed ID11732899
It has been shown that one of the 12 serine residues within the 23 kDa segment of myosin subfragment 1 can be covalently modified with a fluorescent probe 9-anthroylnitrile (ANN) [Hiratsuka, T. (1989) J. Biol. Chem. 264 (30), 18188-18194]. To identify the exact binding site of the probe, the distances ... More
Conserved and variable residues within the Bw4 motif of HLA-B make separable contributions to recognition by the NKB1 killer cell-inhibitory receptor.
AuthorsGumperz JE, Barber LD, Valiante NM, Percival L, Phillips JH, Lanier LL, Parham P
JournalJ Immunol
PubMed ID9164941
Allotypes from four divergent HLA-B families (B8, B15, B16, and B27) were compared for their inhibition of cytolysis by NK cells expressing the NKB1 receptor. Allotypes differing solely at the Bw4/Bw6 region were examined as were a more divergent subset of B15 allotypes. The capacity to interact with NKB1 correlated ... More
Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection.
AuthorsLinder V, Verpoorte E, Thormann W, de Rooij NF, Sigrist H
JournalAnal Chem
PubMed ID11569807
Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization ... More
Detection of protein oxidation in rat-1 fibroblasts by fluorescently labeled tyramine.
Authorsvan der Vlies D, Wirtz KW, Pap EH
JournalBiochemistry
PubMed ID11425304
Oxidative damage to proteins has been postulated as a major cause of various degenerative diseases including the loss of functional capacity during aging. A prominent target for oxidation by reactive oxygen species (ROS) is the tyrosine residue. Here we present a highly sensitive method for the detection of tyrosyl radical ... More
Altered cell surface expression and signaling of leptin receptors containing the fatty mutation.
AuthorsCrouse JA, Elliott GE, Burgess TL, Chiu L, Bennett L, Moore J, Nicolson M, Pacifici RE
JournalJ Biol Chem
PubMed ID9660803
Leptin and the leptin receptor are key players in the regulation of body weight. In an attempt to dissect the molecular mechanism of the Zucker fatty rat leptin receptor mutation (Gln269 --> Pro) we analyzed the effects of this mutation on leptin receptor signaling and expression in three different expression ... More
Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states.
AuthorsMacLean JJ, Chrin LR, Berger CL
JournalBiophys J
PubMed ID10692329
Lys-553 of skeletal muscle myosin subfragment 1 (S1) was specifically labeled with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) and fluorescence quenching experiments were carried out to determine the accessibility of this probe at Lys-553 in both the strongly and weakly actin-bound states of the MgATPase cycle. Solvent ... More
Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry.
In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize ... More
Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.
AuthorsMoore KJ, Turconi S, Ashman S, Ruediger M, Haupts U, Emerick V, Pope AJ
JournalJ Biomol Screen
PubMed ID10838431
Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule ... More
Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements.
AuthorsBene L, Fulwyler MJ, Damjanovich S
JournalCytometry
PubMed ID10918280
BACKGROUND: Perrin equation suggests an alternative way for the accurate energy transfer determination on a cell-by-cell basis by measuring polarized donor intensities in a conventional flow cytometer. METHODS: The relationship between energy transfer and fluorescence anisotropy of the donor was investigated by flow cytometric generation of Perrin-lifetime plots of fluorescent ... More
Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils.
AuthorsCooper WC, Chrin LR, Berger CL
JournalBiophys J
PubMed ID10692330
Myosin subfragment 1 (S1) can be specifically modified at Lys-553 with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) (Bertrand, R., J. Derancourt, and R. Kassab. 1995. Biochemistry. 34:9500-9507), and solvent quenching of FHS-S1 with iodide has been shown to be sensitive to actin binding at low ionic strength ... More
Production and properties of skeletal myosin subfragment 1 selectively labeled with fluorescein at lysine-553 proximal to the strong actin-binding site.
AuthorsBertrand R, Derancourt J, Kassab R
JournalBiochemistry
PubMed ID7626619
We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein. As a ... More
Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells.
Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports ... More
Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1.
AuthorsSmyczynski C, Kasprzak AA
JournalBiochemistry
PubMed ID9341208
The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to ... More
Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies.
AuthorsPrussin C, Metcalfe DD
JournalJ Immunol Methods
PubMed ID8551029
Recently, there have been several reports demonstrating improvements in the flow cytometric detection of intracellular cytokines. These advances, although significant, have not yielded techniques that have easily been translated into broad use. To address this issue, we have coupled a fixation and permeabilization method with the use of directly labelled ... More
Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis.
AuthorsWakarchuk W, Martin A, Jennings MP, Moxon ER, Richards JC
JournalJ Biol Chem
PubMed ID8702594
The biosynthetic function of the lgtABE genetic locus of Neisseria meningitidis was determined by structural analysis of lipopolysaccharide (LPS) derived from mutant strains and enzymic assay for glycosyltransferase activity. LPS was obtained from mutants generated by insertion of antibiotic resistance cassets in each of the three genes lgtA, lgtB, lgtE ... More