We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase ... More
DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.
The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 ... More
Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.
We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding ... More
A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes.
AuthorsBeeton C, Wulff H, Singh S, Botsko S, Crossley G, Gutman GA, Cahalan MD, Pennington M, Chandy KG
JournalJ Biol Chem
PubMed ID12511563
T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells ... More
Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.
AuthorsSmith PR, Saccomani G, Joe EH, Angelides KJ, Benos DJ
JournalProc Natl Acad Sci U S A
PubMed ID1651488
'Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels ... More
Demonstration and analysis of tubulin binding sites on centrosomes.
AuthorsMéda P, Chevrier V, Eddé B, Job D
JournalBiochemistry
PubMed ID9054561
'Microtubule nucleation on centrosomes is vital to the establishment of organized microtubule arrays in cells. Despite recent advances, little is known about the sequence of molecular events which leads to microtubule assembly on centrosomes. A putative early step in the nucleation process is interaction of free tubulin dimers with centrosomes. ... More
Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C).
AuthorsUmeyama T, Okabe S, Kanai Y, Hirokawa N
JournalJ Cell Biol
PubMed ID8421058
'MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed ... More
Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy.
'ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor tyrosine kinase family, is often overexpressed in breast cancer and other malignancies. ErbB2 homodimerizes but also presents as a common auxiliary subunit of the EGF and heregulin receptors (erbB1 or EGFR; and erbB3-4, respectively), with which it heteroassociates. ... More
Evidence that translocation of the proteinase precedes its acylation in the serpin inhibition pathway.
AuthorsMellet P, Bieth JG
JournalJ Biol Chem
PubMed ID10753871
'The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence ... More
pH- and ionic strength-dependent fusion of phospholipid vesicles induced by pardaxin analogues or by mixtures of charge-reversed peptides.
AuthorsRapaport D, Hague GR, Pouny Y, Shai Y
JournalBiochemistry
PubMed ID8461295
'The fusogenic properties of the neurotoxin paradaxin and eight of its analogues with small unilamellar vesicles (SUV), composed of egg phosphatidylcholine and phosphatidylserine (PC/PS), were investigated. Fusion was demonstrated by a lipid-mixing assay and by an increase in vesicle size as revealed by electron microscopy. The lipid-mixing assay was performed ... More
Aggregation and organization of pardaxin in phospholipid membranes. A fluorescence energy transfer study.
AuthorsRapaport D, Shai Y
JournalJ Biol Chem
PubMed ID1551864
'Fluorescence resonance energy transfer has been used to study the aggregation and organization of pardaxin and its analogues within lipid membranes. Peptide molecules labeled with 5- (and 6-) carboxyfluorescein at their N-terminal amino acid served as donors in these energy transfer measurements, whereas peptides labeled with 5- (and 6-) carboxytetramethylrhodamine ... More
The structure and organization of synthetic putative membranous segments of ROMK1 channel in phospholipid membranes.
AuthorsBen-Efraim I, Shai Y
JournalBiophys J
PubMed ID8994595
'The hydropathy plot of ROMK1, an inwardly rectifying K+ channel, suggests that the channel contains two transmembrane domains (M1 and M2) and a linker between them with significant homology to the H5 pore region of voltage-gated K+ channels. To gain structural information on the pore region of the ROMK1 channel, ... More
Control of actin polymerization in live and permeabilized fibroblasts.
AuthorsSymons MH, Mitchison TJ
JournalJ Cell Biol
PubMed ID1860882
'We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin ... More
Structural characterization, membrane interaction, and specific assembly within phospholipid membranes of hydrophobic segments from Bacillus thuringiensis var. israelensis cytolytic toxin.
AuthorsGazit E, Shai Y
JournalBiochemistry
PubMed ID8241124
'The Bacillus thuringiensis var. israelensis (Bti) cytolytic toxin is hypothesized to exert its toxic activity via pore formation in the cell membrane as a result of the aggregation of several monomers. To gain insight into the toxin''s mode of action, 2 putative hydrophobic 22 amino acid peptides were synthesized and ... More
Minimal RNA constructs that specifically bind aminoglycoside antibiotics with high affinities.
AuthorsHamasaki K, Killian J, Cho J, Rando RR
JournalBiochemistry
PubMed ID9425088
'RNA molecules are the functional targets for aminoglycosides. In order to approach an understanding of the rules which underlie aminoglycoside-RNA recognition, high-affinity RNA aptamers have been prepared which discriminate among various aminoglycosides [Wang et al. (1996) Biochemistry 35, 12338-12346]. One of these aptamers, J6, which is 109 nts in length, ... More
Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes.
AuthorsBark SJ, Hahn KM
JournalMethods
PubMed ID10720464
'When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to ... More
A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements.
AuthorsTuschl T, Gohlke C, Jovin TM, Westhof E, Eckstein F
JournalScience
PubMed ID7973630
'For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative ... More
Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan.
AuthorsBeaven AE, Paynter KT
JournalBiol Bull
PubMed ID10065529
'Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH ... More
Region-specific microtubule transport in motile cells.
AuthorsYvon AM, Wadsworth P
JournalJ Cell Biol
PubMed ID11086002
'Photoactivation and photobleaching of fluorescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin-dependent manner, and that the direction of transport ... More
Mode of action of the antibacterial cecropin B2: a spectrofluorometric study.
AuthorsGazit E, Lee WJ, Brey PT, Shai Y
JournalBiochemistry
PubMed ID8075068
'Cecropin B2 (CecB) is a 35 amino acid residue, antibacterial peptide that was isolated from the hemolymph and cuticular matrix of the silkworm, Bombyx mori. Synthetic peptides with sequences corresponding to CecB and its truncated analogue, [3-->35]CecB, were synthesized and selectively labeled at their N-terminal amino acids with either 7-nitrobenz-2-oxa-1,3-diazol-4-yl ... More
Enolase exists in the fluid phase of cytoplasm in 3T3 cells.
AuthorsPagliaro L, Kerr K, Taylor DL
JournalJ Cell Sci
PubMed ID2621228
'We have investigated the intracellular distribution and mobility of the glycolytic enzyme enolase, using functional fluorescent analogs labeled with the succinimidyl esters of carboxyfluorescein (F1-enolase) and carboxytetramethylrhodamine (Rh-enolase) In contrast to aldolase, neither native enolase nor labeled enolase gelled filamentous actin (F-actin), as measured by falling-ball viscometry, indicating a lack ... More
Direct measurement of thermodynamic and kinetic parameters of DNA triple helix formation by fluorescence spectroscopy.
AuthorsYang M, Ghosh SS, Millar DP
JournalBiochemistry
PubMed ID7803396
'Direct measurement of thermodynamic and kinetic parameters of oligonucleotide-directed DNA triple helix formation has been achieved by fluorescence spectroscopic methods. Fluorescence resonance energy transfer (FRET) was used to study the binding of an acceptor-labeled single-stranded oligonucleotide to a donor-labeled DNA duplex. Equilibrium binding constants and association rate constants for triplex ... More
Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes.
AuthorsVámosi G, Gohlke C, Clegg RM
JournalBiophys J
PubMed ID8842236
'Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5'' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the ... More
Cutting edge: egress of newly generated plasma cells from peripheral lymph nodes depends on beta 2 integrin.
AuthorsPabst O, Peters T, Czeloth N, Bernhardt G, Scharffetter-Kochanek K, Förster R
JournalJ Immunol
PubMed ID15944247
'During humoral immune responses, naive B cells differentiate into Ab-secreting plasma cells within secondary lymphoid organs. Differentiating plasma cells egress from their sites of generation and redistribute to other tissues, predominantly the bone marrow and mucosal tissues. In this study, we demonstrate that within peripheral lymph nodes newly generated plasma ... More
In situ visualization of caspase-1-like activity associated with promotion of hippocampal cell death.
AuthorsNishii W, Shoda T, Matsumoto N, Nakamura T, Kudo Y, Takahashi K
JournalFEBS Lett
PubMed ID11997036
'To clarify the function of caspase-1-like proteases in neuronal cell death, it is important to be able to detect the activity in living organs by microscopic visualization. In the present study, we synthesized a novel fluorescent substrate sensitive to the caspase-1-like activity, which is easily introduced into cells constituting living ... More
TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts.
AuthorsO'Brien LL, Wiese C
JournalJ Cell Biol
PubMed ID16735579
'Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in ... More
Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.
AuthorsJu J, Ruan C, Fuller CW, Glazer AN, Mathies RA
JournalProc Natl Acad Sci U S A
PubMed ID7753809
'Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5'' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within ... More
Synthetic S-2 and H-5 segments of the Shaker K+ channel: secondary structure, membrane interaction, and assembly within phospholipid membranes.
AuthorsPeled H, Shai Y
JournalBiochemistry
PubMed ID8003486
'Current models of voltage-activated K+ channels predict that the channels are formed by the coassembly of four polypeptide monomers, each of which consists of six transmembrane segments (S1-S6) and long terminal domains. The aqueous pores are thought to be composed of the conserved H-5 regions contributed by four monomers. In ... More
Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides.
AuthorsTong AK, Ju J
JournalNucleic Acids Res
PubMed ID11861924
'Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide ... More
Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis.
AuthorsGhosh JK, Shaool D, Guillaud P, Cicéron L, Mazier D, Kustanovich I, Shai Y, Mor A
JournalJ Biol Chem
PubMed ID9395500
'The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. ... More
Microtubule behavior in PC12 neurites: variable results obtained with photobleach technology.
AuthorsKeith CH, Farmer MA
JournalCell Motil Cytoskeleton
PubMed ID8402955
'We have examined the effects of various means of photobleaching on the recovery of fluorescence, movement, and morphology of the microtubules in the neurites of rhodamine-tubulin-injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different ... More
A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.
AuthorsUrdea MS, Warner BD, Running JA, Stempien M, Clyne J, Horn T
JournalNucleic Acids Res
PubMed ID3387214
'N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5''-O-dimethoxy trityl -5-methyl-2''-deoxycytidine-3''-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels ... More
Coiled-coil tag--probe system for quick labeling of membrane receptors in living cell.
AuthorsYano Y, Yano A, Oishi S, Sugimoto Y, Tsujimoto G, Fujii N, Matsuzaki K,
JournalACS Chem Biol
PubMed ID18533657
'The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of ... More
Localization and mobility of omega-conotoxin-sensitive Ca2+ channels in hippocampal CA1 neurons.
AuthorsJones OT, Kunze DL, Angelides KJ
JournalScience
PubMed ID2543080
'Voltage-dependent Ca2+ channels (VDCCs) are modulators of synaptic plasticity, oscillatory behavior, and rhythmic firing in brain regions such as the hippocampus. The distribution and lateral mobility of VDCCs on CA1 hippocampal neurons have been determined with biologically active fluorescent and biotinylated derivatives of the selective probe omega-conotoxin in conjunction with ... More
Microtubule behavior in the growth cones of living neurons during axon elongation.
AuthorsTanaka EM, Kirschner MW
JournalJ Cell Biol
PubMed ID1918145
'To understand how microtubules are generated in the growth cone, we have imaged fluorescently tagged microtubules in living frog embryonic neurons. The neurons were labeled by injecting rhodamine-labeled tubulin into the fertilized egg and explanting the neurons from the neural tube. Microtubules extend deep into the growth cone periphery and ... More
Membrane interaction and self-assembly within phospholipid membranes of synthetic segments corresponding to the H-5 region of the shaker K+ channel.
AuthorsPeled H, Shai Y
JournalBiochemistry
PubMed ID8347593
'The voltage-activated K+ channels are assumed to be formed by the coassembly of four polypeptide monomers. Each of these monomers is postulated to consist of six transmembrane segments (S1 to S6), and long N- and C-terminal domains. The highly conserved linker, H-5, between the fifth and the sixth transmembrane segments, ... More
Detection and analysis of tumor fluorescence using a two-photon optical fiber probe.
AuthorsThomas TP, Myaing MT, Ye JY, Candido K, Kotlyar A, Beals J, Cao P, Keszler B, Patri AK, Norris TB, Baker JR
JournalBiophys J
PubMed ID15189892
'The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing ... More
Ribozymes as tools for therapeutic target validation in arthritis.
AuthorsJarvis TC, Bouhana KS, Lesch ME, Brown SA, Parry TJ, Schrier DJ, Hunt SW, Pavco PA, Flory CM
JournalJ Immunol
PubMed ID10861088
'In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to ... More
Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins.
'The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free ... More
Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region.
AuthorsWang Y, Hamasaki K, Rando RR
JournalBiochemistry
PubMed ID9020774
'Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation ... More
Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells.
AuthorsFisher TL, Terhorst T, Cao X, Wagner RW
JournalNucleic Acids Res
PubMed ID8396239
'The intracellular distribution and metabolism of microinjected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, microinjected into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15-20 minutes. Microinjected fluorescent phosphorothioate ODNs remain in the nucleus for ... More
Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels.
AuthorsDaban JR, Bartolomé S, Samsó M
JournalAnal Biochem
PubMed ID1725949
'In a previous work (J.-R. Daban, M. Samsó, and S. Bartolomé, Anal. Biochem. 199, 162-168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[alpha]-phenoxazine-5-one (Nile red). This enhancement ... More
Evidence for assembly of small multidrug resistance proteins by a "two-faced" transmembrane helix.
AuthorsRath A, Melnyk RA, Deber CM
JournalJ Biol Chem
PubMed ID16574656
'Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the ... More
Formation of filopodia-like bundles in vitro from a dendritic network.
'We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) ... More
A new class of homogeneous nucleic acid probes based on specific displacement hybridization.
AuthorsLi Q, Luan G, Guo Q, Liang J
JournalNucleic Acids Res
PubMed ID11788731
'We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but ... More
Single fungal kinesin motor molecules move processively along microtubules.
AuthorsLakämper S, Kallipolitou A, Woehlke G, Schliwa M, Meyhöfer E
JournalBiophys J
PubMed ID12609885
'Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in ... More
Synthesis and pharmacological activity of fluorescent histamine H1 receptor antagonists related to mepyramine.
AuthorsLi L, Kracht J, Peng S, Bernhardt G, Buschauer A
JournalBioorg Med Chem Lett
PubMed ID12657255
'Fluorescently labeled histamine H(1) receptor antagonists were synthesized starting from N-demethylmepyramine by introduction of omega-aminoalkyl chains (2-8 methylene groups in length) followed by derivatization of the terminal NH(2) group with various fluorophores (fluorescein, naphthofluorescein, rhodamine, tetramethylrhodamine, BODIPY, dansyl, and nitrobenzoxadiazole (NBD)). On the isolated guinea pig ileum and in a ... More
Extending the applicability of carboxyfluorescein in solid-phase synthesis.
AuthorsFischer R, Mader O, Jung G, Brock R
JournalBioconjug Chem
PubMed ID12757391
'Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing postsynthesis workup. For the cost-efficient labeling of large compound ... More
Ultra-high-speed DNA sequencing using capillary electrophoresis chips.
AuthorsWoolley AT, Mathies RA
JournalAnal Chem
PubMed ID8644919
'DNA sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 microns cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA sequencing fragment ladders were produced and fluorescently ... More
Real-time PCR in the microbiology laboratory.
AuthorsMackay IM
JournalClin Microbiol Infect
PubMed ID15008940
'Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has ... More
Synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 for use in the assay of alpha-glucosidase I activity.
AuthorsScaman CH, Hindsgaul O, Palcic MM, Srivastava OP
JournalCarbohydr Res
PubMed ID9008849
'The chemical synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp p-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 (9), a substrate specific for alpha-glucosidase I, is reported. This enzyme removes the terminal alpha-D-Glcp unit to produce alpha-D-Glcp-(1-->3)-alpha-D-Glcp-O-(CH2)8 COOCH3 (10). This is the first synthetic substrate described for glucosidase I that allows kinetic evaluation of substrates and inhibitors of this enzyme. ... More
Optimizing primer--probe design for fluorescent PCR.
AuthorsProudnikov D, Yuferov V, Zhou Y, LaForge KS, Ho A, Kreek MJ
JournalJ Neurosci Methods
PubMed ID12581847
'TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then ... More
Optical properties and application of a reactive and bioreducible thiol-containing tetramethylrhodamine dimer.
'Thiolated dimeric tetramethylrhodamine (TAMRA) was synthesized in a straightforward procedure utilizing commercially available 5(6)-succinimidyl TAMRA and cystamine hydrochloride. The thiol-containing TAMRA dimer displayed distinct spectral properties in reduced and oxidized forms; covalent dimer formation produced greater effects on the spectral properties than previously reported for noncovalent TAMRA dimers or dimers ... More
A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents.
AuthorsBrinkley M
JournalBioconjug Chem
PubMed ID1616945
H-type dimer formation of fluorophores: a mechanism for activatable, in vivo optical molecular imaging.
AuthorsOgawa M, Kosaka N, Choyke PL, Kobayashi H,
JournalACS Chem Biol
PubMed ID19480464
'In vivo molecular imaging with target-specific activatable '
Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment.
AuthorsFurey WS, Joyce CM, Osborne MA, Klenerman D, Peliska JA, Balasubramanian S
JournalBiochemistry
PubMed ID9485450
'Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I. Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors ... More
Fluorescent microtubules break up under illumination.
AuthorsVigers GP, Coue M, McIntosh JR
JournalJ Cell Biol
PubMed ID3417772
'We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show ... More
Enhanced FTY720-mediated lymphocyte homing requires G alpha i signaling and depends on beta 2 and beta 7 integrin.
AuthorsPabst O, Herbrand H, Willenzon S, Worbs T, Schippers A, Müller W, Bernhardt G, Förster R
JournalJ Immunol
PubMed ID16424175
'The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this ... More
Mechanism and dynamics of breakage of fluorescent microtubules.
AuthorsGuo H, Xu C, Liu C, Qu E, Yuan M, Li Z, Cheng B, Zhang D
JournalBiophys J
PubMed ID16387782
'The breakage of fluorescence-labeled microtubules under irradiation of excitation light is found in our experiments. Its mechanism is studied. The results indicate that free radicals are the main reason for the photosensitive breakage. Furthermore, the mechanical properties of the microtubules are probed with a dual-optical tweezers system. It is found ... More
A peptide derived from a conserved domain of Sendai virus fusion protein inhibits virus-cell fusion. A plausible mode of action.
AuthorsGhosh JK, Shai Y
JournalJ Biol Chem
PubMed ID9516418
'SV-201, a peptide derived from a conserved and potentially amphipathic region (amino acids 201-229) in the Sendai virus ectodomain, specifically inhibited virus-mediated hemolysis only when added to virions prior to their attachment to red blood cells. Sendai virus-mediated hemagglutinin assay in the presence of SV-201 demonstrated that the peptide does ... More
Transferrin receptor containing the SDYQRL motif of TGN38 causes a reorganization of the recycling compartment but is not targeted to the TGN.
AuthorsJohnson AO, Ghosh RN, Dunn KW, Garippa R, Park J, Mayor S, Maxfield FR, McGraw TE
JournalJ Cell Biol
PubMed ID8991088
'The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter ... More
Clustering and mobility of voltage-dependent sodium channels during myelination.
AuthorsJoe EH, Angelides KJ
JournalJ Neurosci
PubMed ID8392537
'In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quick, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control ... More
Structural and kinetic studies of a cisplatin-modified DNA icosamer binding to HMG1 domain B.
AuthorsJamieson ER, Jacobson MP, Barnes CM, Chow CS, Lippard SJ
JournalJ Biol Chem
PubMed ID10212205
'The high mobility group (HMG) domain is a DNA-binding motif found in the non-histone chromosomal proteins, HMG1 and HMG2, and some transcription factors. Experimental evidence has demonstrated that HMG-domain proteins can play a role in sensitizing cells to the anticancer drug cisplatin. Fluorescence resonance energy transfer (FRET) experiments were performed ... More
Interaction of the mammalian antibacterial peptide cecropin P1 with phospholipid vesicles.
AuthorsGazit E, Boman A, Boman HG, Shai Y
JournalBiochemistry
PubMed ID7547876
'Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity ... More
Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP.
AuthorsHiguchi H, Muto E, Inoue Y, Yanagida T
JournalProc Natl Acad Sci U S A
PubMed ID9114000
'To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension ... More
Axonal transport of tubulin in Ti1 pioneer neurons in situ.
AuthorsSabry J, O'Connor TP, Kirschner MW
JournalNeuron
PubMed ID7541635
'In neurons, tubulin is synthesized only in the cell body or dendrites, yet the growing axon requires a steady supply of this protein at the growth cone. Hence, some mechanism must exist to move tubulin from the cell body to the growth cone. Transport could conceivably occur by simple diffusion, ... More
Quantitative fluorescence techniques for the determination of local microtubule polymerization equilibria in cultured neurons.
AuthorsKeith CH
JournalJ Neurosci Methods
PubMed ID1798344
'The local control of intracellular microtubule polymerization equilibria has been hypothesized to be an important factor in the determination of neurite extension and other examples of cellular asymmetry. Provided that the quantum yield of the fluorophore remains constant, the combination of fluorescent analogue cytochemistry, differential extraction protocols, and quantitative video ... More
Structure-function study of a heptad repeat positioned near the transmembrane domain of Sendai virus fusion protein which blocks virus-cell fusion.
AuthorsGhosh JK, Peisajovich SG, Ovadia M, Shai Y
JournalJ Biol Chem
PubMed ID9765238
'A synthetic heptad repeat, SV-473, derived from Sendai virus fusion protein is a potent inhibitor of virus-cell fusion. In order to understand the mechanism of the inhibitory effect, we synthesized and fluorescently labeled SV-465, an extended version of SV-473 by one more heptad, its mutant peptide A17,24-SV-465, in which two ... More
Distribution and lateral mobility of voltage-dependent sodium channels in neurons.
AuthorsAngelides KJ, Elmer LW, Loftus D, Elson E
JournalJ Cell Biol
PubMed ID2454930
'Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltage-dependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which ... More
Energy-transfer fluorescent reagents for DNA analyses.
AuthorsGlazer AN, Mathies RA
JournalCurr Opin Biotechnol
PubMed ID9013654
'Fluorescence resonance energy transfer has facilitated the development of a new class of high-performance fluorescent labeling reagents for multiplex analyses of nucleic acids. The enhanced emission of energy transfer (ET) primers has provided a decadic improvement in the performance of automated DNA sequencers. The emission spectral purity of ET primers ... More
Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.
AuthorsWelch MD, Rosenblatt J, Skoble J, Portnoy DA, Mitchison TJ
JournalScience
PubMed ID9651243
'Actin filament assembly at the cell surface of the pathogenic bacterium Listeria monocytogenes requires the bacterial ActA surface protein and the host cell Arp2/3 complex. Purified Arp2/3 complex accelerated the nucleation of actin polymerization in vitro, but pure ActA had no effect. However, when combined, the Arp2/3 complex and ActA ... More
Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors.
AuthorsHubl U, Slim GC, Zubkova OV
JournalAnal Biochem
PubMed ID10998267
'Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) ... More
Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.
AuthorsClegg RM, Murchie AI, Zechel A, Lilley DM
JournalProc Natl Acad Sci U S A
PubMed ID8464916
'The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5'' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of double-stranded ... More
The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex.
AuthorsGazit E, Sauer RT
JournalJ Biol Chem
PubMed ID10358024
'The toxin (Doc) and antidote (Phd) proteins of the plasmid addiction system of bacteriophage P1 were purified as a complex. Cocrystals of the complex contained a 2:1 molar ratio of Phd:Doc as assayed by dye binding following SDS-polyacrylamide gel electrophoresis and as determined by amino acid analysis. Gel filtration and ... More
A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.
AuthorsStanton LW, White RT, Bryant CM, Protter AA, Endemann G
JournalJ Biol Chem
PubMed ID1429595
'The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL ... More
Energy transfer primers with 5- or 6-carboxyrhodamine-6G as acceptor chromophores.
AuthorsHung SC, Ju J, Mathies RA, Glazer AN
JournalAnal Biochem
PubMed ID8660606
'Energy-transfer (ET) fluorescent primers for DNA sequencing and multiplex genetic analysis (Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351) are named according to the convention D-N-A, where D is the donor, N is the number of ... More
Labelling peptides with fluorescent probes for incorporation into degradable polymers.
AuthorsBrunner A, Minamitake Y, Göpferich A
JournalEur J Pharm Biopharm
PubMed ID9653631
'Two peptides, atrial natriuretic peptide (ANP) and salmon calcitonin (sCT) were conjugated with a fluorescent, amine-reactive probe 5-(and 6-)carboxytetramethylrhodamine,-succinimidylester (5-(6)-TAMRA-SE). The labelling reaction was followed by HPLC and found to be complete after 2 h. The labelled peptides were purified by gel filtration chromatography and characterised by [1H]NMR, UV/VIS and ... More
Automated velocity programming for increased detection zone residence times in capillary electrophoresis.
AuthorsShear JB, Colón LA, Zare RN
JournalAnal Chem
PubMed ID7906105
Fluorescence assays to study structure, dynamics, and function of RNA and RNA-ligand complexes.
AuthorsWalter NG, Burke JM
JournalMethods Enzymol
PubMed ID10829293
Metabolic cytometry: monitoring oligosaccharide biosynthesis in single cells by capillary electrophoresis.
AuthorsKrylov SN, Arriaga EA, Chan NW, Dovichi NJ, Palcic MM
JournalAnal Biochem
PubMed ID10906232
Fluorescence excitation transfer immunoassay (FETI).
AuthorsUllman EF, Khanna PL
JournalMethods Enzymol
PubMed ID7033726
Triple fluorescence energy transfer in covalently trichromophore-labeled DNA.
AuthorsTong AK, Jockusch S, Li Z, Zhu HR, Akins DL, Turro NJ, Ju J
JournalJ Am Chem Soc
PubMed ID11749560
Simultaneous detection of TaqMan probes containing Fam and Tamra reporter fluorophores.
AuthorsNasarabadi S, Milanovich F, Richards J, Belgrader P
JournalBiotechniques
PubMed ID10631486
4',5'-Dimethoxy-6-carboxyfluorescein: a novel dipole-dipole coupled fluorescence energy transfer acceptor useful for fluorescence immunoassays.
AuthorsKhanna PL, Ullman EF
JournalAnal Biochem
PubMed ID7006452
A DNA assay based on fluorescence resonance energy transfer and DNA triplex formation.
AuthorsYang M, Ren LQ, Huang M, Kong RY, Fong WF
JournalAnal Biochem
PubMed ID9618207
Recent advances in capillary electrophoresis of carbohydrates.
AuthorsEl Rassi Z, Mechref Y
JournalElectrophoresis
PubMed ID8900934
Preparation of modified tubulins.
AuthorsHyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T
JournalMethods Enzymol
PubMed ID2034137
A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin.
AuthorsMichelot A, Derivery E, Paterski-Boujemaa R, Guérin C, Huang S, Parcy F, Staiger CJ, Blanchoin L,
JournalCurr Biol
PubMed ID17027489
Actin-filament bundles (or cables) have a structural role during cell division and morphogenesis, but also serve as important
Dual-fluorophore ratiometric pH nanosensor with tuneable pK(a) and extended dynamic range.
AuthorsChauhan VM, Burnett GR, Aylott JW,
JournalAnalyst
PubMed ID21416087
Ratiometric pH nanosensors with tuneable pK(a) were prepared by entrapping combinations of two pH-sensitive fluorophores (fluorescein isothiocyanate dextran (FITC-D) and Oregon Green(®) dextran (OG-D)) and a reference fluorophore (5-(and-6)-carboxytetramethylrhodamine dextran (TAMRA-D)), in a biocompatible polymer matrix. Dual-fluorophore pH nanosensors permit the measurement of an extended dynamic range, from pH 4.0 ... More
Protein quantitation in 2-D gels using fluorescence with water Raman as an internal standard.
We have developed a method to enhance fluorescence quantification in two-dimensional gel electrophoresis using the inherent Raman scattering of water as an internal standard. We demonstrate this water internal standard (WIS) method using quantitative comparisons of commercially available protein standards that were either covalently tagged or passively stained with fluorescent ... More
High-density fiber-optic DNA random microsphere array.
AuthorsFerguson JA, Steemers FJ, Walt DR
JournalAnal Chem
PubMed ID11101240
A high-density fiber-optic DNA microarray sensor was developed to monitor multiple DNA sequences in parallel. Microarrays were prepared by randomly distributing DNA probe-functionalized 3.1-microm-diameter microspheres in an array of wells etched in a 500-microm-diameter optical imaging fiber. Registration of the microspheres was performed using an optical encoding scheme and a ... More
Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts.
AuthorsVale RD
JournalCell
PubMed ID1671762
Eukaryotic cells disassemble and reorganize their cytoskeleton during the cell cycle and in response to environmental cues. Disassembly of the actin cytoskeleton is aided by proteins that sever filamentous actin, but microtubule-severing proteins thus far have not been identified. Here, we describe an activity in extracts from Xenopus eggs that ... More
Multiplex capillary denaturing high-performance liquid chromatography with laser-induced fluorescence detection.
AuthorsXiao W, Stern D, Jain M, Huber CG, Oefner PJ
JournalBiotechniques
PubMed ID11414227
Denaturing high-performance liquid chromatography (DHPLC) is a sensitive, robust, and operationally inexpensive method for the detection of single-base substitutions and small deletions and insertions. To increase sample throughout, we have developed a multiplexing strategy using fluorophores to distinguish different PCR products. The system combines recent advances in the synthesis of ... More
Separation of aminated monosaccharides by capillary zone electrophoresis with laser-induced fluorescence detection.
AuthorsZhao JY, Diedrich P, Zhang Y, Hindsgaul O, Dovichi NJ
JournalJ Chromatogr B Biomed Appl
PubMed ID7952095
The six most abundant hexoses found in mammalian carbohydrates were derivatized to serve as standards for an oligosaccharide sequencing project. Glucose, galactose, mannose, and fucose were reductively aminated to produce 1-amino-1-deoxy alditols. Standards for N-acetylglucosamine and N-acetylgalactosamine were synthesized by reducing glucosamine and galactosamine to 2-amino-2-deoxy alditols. The monosaccharides were ... More
Global structure of three-way DNA junctions with and without additional unpaired bases: a fluorescence resonance energy transfer analysis.
The structure of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges with n = 0, 1, 2, and 3) has been investigated by fluorescence resonance energy transfer. The structure of the junction without bulged nucleotides was ... More
A chemical method for fast and sensitive detection of DNA synthesis in vivo.
AuthorsSalic A, Mitchison TJ,
JournalProc Natl Acad Sci U S A
PubMed ID18272492
We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in ... More
Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin.
AuthorsGruber HJ, Marek M, Schindler H, Kaiser K
JournalBioconjug Chem
PubMed ID9258455
Conventional biotin-fluorophore conjugates with approximately 14 atom spacers lose most of their fluorescence when binding to avidin or streptavidin, as is demonstrated in the present study. This explains the unusual fact that only biotinylated marker enzymes, but not fluorescent biotins, are regularly used in bioanalytic assays. Novel biotin-spacer-fluorophore conjugates are ... More
Detection of carbohydrates in capillary electrophoresis.
AuthorsPaulus A, Klockow A
JournalJ Chromatogr A
PubMed ID8601201
This review focuses on recent developments in sensitive detection modes for carbohydrates after separation by capillary electrophoretic methods. To bring detection sensitivity for carbohydrates analysis in line with current methods in protein sequencing, concentration detection limits of 10(-6) molar or better are required. A discussion of mass detection limits and ... More
Bidirectional translocation of neurofilaments along microtubules mediated in part by dynein/dynactin.
Neuronal cytoskeletal elements such as neurofilaments, F-actin, and microtubules are actively translocated by an as yet unidentified mechanism. This report describes a novel interaction between neurofilaments and microtubule motor proteins that mediates the translocation of neurofilaments along microtubules in vitro. Native neurofilaments purified from spinal cord are transported along microtubules ... More