Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans.
AuthorsSandgren S, Cheng F, Belting M
JournalJ Biol Chem
PubMed ID12163493
'New therapies based on gene transfer and protein delivery require a better understanding of the basic mechanisms of macromolecular membrane transport. We have studied cellular uptake of macromolecular polyanions, i.e. DNA and glycosaminoglycans, and a polybasic HIV-Tat derived peptide (GRKKRRQRRRPPQC) using fluorescence assisted cell sorting and confocal fluorescence microscopy. The ... More
A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells.
AuthorsHama Y, Urano Y, Koyama Y, Bernardo M, Choyke PL, Kobayashi H
JournalBioconjug Chem
PubMed ID17105220
'In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away ... More
Activation of murine macrophages by Neisseria meningitidis and IFN-gamma in vitro: distinct roles of class A scavenger and Toll-like pattern recognition receptors in selective modulation of surface phenotype.
AuthorsMukhopadhyay S, Peiser L, Gordon S
JournalJ Leukoc Biol
PubMed ID15218052
'Innate and adaptive immune activation of macrophages (Mphi) by microorganisms and antigen-activated lymphoid cells, respectively, plays an important role in host defense and immunopathology. Antigen-presenting cells express a range of pattern recognition receptors including the class A types I and II scavenger receptors (SR-A) and Toll-like receptors (TLR). Recognition of ... More
Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets.
AuthorsBieschke J, Giese A, Schulz-Schaeffer W, Zerr I, Poser S, Eigen M, Kretzschmar H
JournalProc Natl Acad Sci U S A
PubMed ID10805803
'A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrP(Sc)). However, no test for PrP(Sc) in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule ... More
Intracellular calmodulin availability accessed with two-photon cross-correlation.
AuthorsKim SA, Heinze KG, Waxham MN, Schwille P
JournalProc Natl Acad Sci U S A
PubMed ID14695888
'The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca(2+) signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent ... More
Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy.
AuthorsHess ST, Webb WW
JournalBiophys J
PubMed ID12324447
'Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 micros to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation ... More
Effects of gel material on fluorescence lifetime detection of dyes and dye-labeled DNA primers in capillary electrophoresis.
AuthorsLi L, McGown LB
JournalJ Chromatogr A
PubMed ID10360329
'Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. ... More
Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics.
AuthorsHeinze KG, Rarbach M, Jahnz M, Schwille P
JournalBiophys J
PubMed ID12202390
'Dual-color fluorescence cross-correlation analysis is a powerful tool for probing interactions of different fluorescently labeled molecules in aqueous solution. The concept is the selective observation of coordinated spontaneous fluctuations in two separate detection channels that unambiguously reflect the existence of physical or chemical linkages among the different fluorescent species. It ... More
DNA sequencing by capillary electrophoresis with four-decay fluorescence detection.
AuthorsHe H, McGown LB
JournalAnal Chem
PubMed ID11140750
'A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and ... More
Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum.
AuthorsNeuhaus EM, Almers W, Soldati T
JournalMol Biol Cell
PubMed ID11950947
'Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is ... 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
Natural communities of novel archaea and bacteria with a string-of-pearls-like morphology: molecular analysis of the bacterial partners.
AuthorsMoissl C, Rudolph C, Huber R
JournalAppl Environ Microbiol
PubMed ID11823239
'A recently discovered bacterial/archaeal association, growing in a string-of-pearls-like structure, thrives in the cold (approximately 10 degrees C) sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. It forms characteristic, macroscopically visible globules, the pearls, containing microcolonies of novel euryarchaeota, which are surrounded by mainly filamentous bacteria (C. ... More
Determination of optimal rhodamine fluorophore for in vivo optical imaging.
AuthorsLongmire MR, Ogawa M, Hama Y, Kosaka N, Regino CA, Choyke PL, Kobayashi H,
JournalBioconjug Chem
PubMed ID18610943
'Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high ... More
Ligand-induced internalization of cholecystokinin receptors. Demonstration of the importance of the carboxyl terminus for ligand-induced internalization of the rat cholecystokinin type B receptor but not the type A receptor.
AuthorsPohl M, Silvente-Poirot S, Pisegna JR, Tarasova NI, Wank SA
JournalJ Biol Chem
PubMed ID9218453
'Internalization of a variety of different heptahelical G protein-coupled receptors has been shown to be influenced by a number of different structural determinants of the receptors, including the carboxyl terminus. To investigate the role of the carboxyl terminus of cholecystokinin (CCK) receptors in receptor internalization, the rat wild type (WT) ... More
Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy.
AuthorsKunst BH, Schots A, Visser AJ
JournalAnal Chem
PubMed ID12403592
'Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary ... More
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 '
PCR-free detection of genetically modified organisms using magnetic capture technology and fluorescence cross-correlation spectroscopy.
AuthorsZhou X, Xing D, Tang Y, Chen WR,
JournalPLoS One
PubMed ID19956680
'The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a ... 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
Frequency and voltage dependence of the dielectrophoretic trapping of short lengths of DNA and dCTP in a nanopipette.
AuthorsYing L, White SS, Bruckbauer A, Meadows L, Korchev YE, Klenerman D
JournalBiophys J
PubMed ID14747337
'The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide ... More
Evaluation of the phosphorescent palladium(II)-coproporphyrin labels in separation-free hybridization assays.
AuthorsBurke M, O'Sullivan PJ, Soini AE, Berney H, Papkovsky DB
JournalAnal Biochem
PubMed ID12927834
'Palladium(II)-coproporphyrin label and a set of corresponding monofunctional labeling reagents with different linker arms were evaluated for labeling of oligonucleotides and subsequent use in hybridization assays. The properties of resulting oligonucleotide probes including phosphorescence spectra, quantum yields, lifetimes, and labeling yields were examined as functions of the label and oligonucleotide ... More
Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy.
AuthorsChattopadhyay K, Saffarian S, Elson EL, Frieden C
JournalProc Natl Acad Sci U S A
PubMed ID12381795
'Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel beta-strands enclosing a large ... More
Programmable delivery of DNA through a nanopipet.
AuthorsYing L, Bruckbauer A, Rothery AM, Korchev YE, Klenerman D
JournalAnal Chem
PubMed ID11922307
We report the pulsed delivery of single-stranded DNA molecules through a nanopipet. The conical geometry of the pipet leads to a localized electric field, since all of the potential drop occurs in the tip region. Pulsatile delivery of DNA molecules can be achieved in an experimentally simple way with high ... More
Ultrasensitive coincidence fluorescence detection of single DNA molecules.
AuthorsLi H, Ying L, Green JJ, Balasubramanian S, Klenerman D
JournalAnal Chem
PubMed ID12705600
We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly ... More
Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.
AuthorsDoi N, Takashima H, Kinjo M, Sakata K, Kawahashi Y, Oishi Y, Oyama R, Miyamoto-Sato E, Sawasaki T, Endo Y, Yanagawa H
JournalGenome Res
PubMed ID11875038
We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, ... More
Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates.
AuthorsGiller G, Tasara T, Angerer B, Mühlegger K, Amacker M, Winter H
JournalNucleic Acids Res
PubMed ID12736313
Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In ... More
Spectral fluorescence molecular imaging of lung metastases targeting HER2/neu.
AuthorsKoyama Y, Hama Y, Urano Y, Nguyen DM, Choyke PL, Kobayashi H,
JournalClin Cancer Res
PubMed ID17504994
PURPOSE: Surgical resection of pulmonary metastases is now a clinically accepted cancer therapy but its success depends on the accurate localization and removal of all tumor foci. To enhance the detection of pulmonary metastases during surgery, we developed an i.v. administered optical probe that uses a monoclonal antibody, Herceptin (trastuzumab), ... More
Gene expression analysis using single molecule detection.
AuthorsKorn K, Gardellin P, Liao B, Amacker M, Bergström A, Björkman H, Camacho A, Dörhöfer S, Dörre K, Enström J, Ericson T, Favez T, Gösch M, Honegger A, Jaccoud S, Lapczyna M, Litborn E, Thyberg P, Winter H, Rigler R
JournalNucleic Acids Res
PubMed ID12907741
Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two ... More
Comparison of fluorescence energy transfer primers with different donor-acceptor dye combinations.
AuthorsHung SC, Mathies RA, Glazer AN
JournalAnal Biochem
PubMed ID9448839
Fluorescence energy transfer (ET) primers are far superior to single dye-labeled primers as labels for DNA sequencing and polymerase chain reaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor fluorescence emission intensity and the amount of residual donor fluorescence ... More
Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.
AuthorsSchwille P, Meyer-Almes FJ, Rigler R
JournalBiophys J
PubMed ID9083691
The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as ... More
DNA analysis by fluorescence quenching detection.
AuthorsXiao M, Kwok PY
JournalGenome Res
PubMed ID12727909
The analysis of human genetic variations such as single nucleotide polymorphisms (SNPs) has great applications in genome-wide association studies of complex genetic traits. We have developed an SNP genotyping method based on the primer extension assay with fluorescence quenching as the detection. The template-directed dye-terminator incorporation with fluorescence quenching detection ... More
Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA.
AuthorsTasara T, Angerer B, Damond M, Winter H, Dörhöfer S, Hübscher U, Amacker M
JournalNucleic Acids Res
PubMed ID12736314
The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified ... More
A high-throughput assay for Tn5 Tnp-induced DNA cleavage.
AuthorsAson B, Reznikoff WS
JournalNucleic Acids Res
PubMed ID15201394
Transposition causes genomic instability by mobilizing DNA elements. This phenomenon is mechanistically related to other DNA rearrangements, such as V(D)J recombination and retroviral DNA integration. A conserved active site architecture within the transposase/integrase superfamily catalyzes these distinct phenomena. The Tn5 transposase (Tnp) falls within this protein class, and many intermediates ... More
Expression of fragments of translation initiation factor eIF4GI reveals a nuclear localisation signal within the N-terminal apoptotic cleavage fragment N-FAG.
AuthorsColdwell MJ, Hashemzadeh-Bonehi L, Hinton TM, Morley SJ, Pain VM
JournalJ Cell Sci
PubMed ID15128869
The eukaryotic initiation factor eIF4GI plays a central role in the assembly of a competent initiation complex at the 5' end of an mRNA. Five isoforms of eIF4G exist in cells, arising from alternative translation initiation. During picornaviral infection or apoptosis, eIF4GI is cleaved proteolytically to yield distinct fragments. Using ... More
In situ analysis of single-stranded and duplex siRNA integrity in living cells.
AuthorsRaemdonck K, Remaut K, Lucas B, Sanders NN, Demeester J, De Smedt SC
JournalBiochemistry
PubMed ID16939213
To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe ... More
Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing.
AuthorsNunnally BK, He H, Li LC, Tucker SA, McGown LB
JournalAnal Chem
PubMed ID9212706
Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of ... More
Binding of a fluorescent lipid amphiphile to albumin and its transfer to lipid bilayer membranes.
AuthorsAbreu MS, Estronca LM, Moreno MJ, Vaz WL
JournalBiophys J
PubMed ID12524292
Kinetics and thermodynamics of the binding of a fluorescent lipid amphiphile, Rhodamine Green(TM)-tetradecylamide (RG-C(14:0)), to bovine serum albumin were characterized in an equilibrium titration and by stopped-flow fluorimetry. The binding equilibrium of RG-C(14:0) to albumin was then used to reduce its concentration in the aqueous phase to a value below ... More
Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations.
AuthorsTonon G, Roschke A, Stover K, Shou Y, Kuehl WM, Kirsch IR
JournalGenes Chromosomes Cancer
PubMed ID10719373
Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization ... More
Dynamic behavior of rod photoreceptor disks.
AuthorsChen C, Jiang Y, Koutalos Y
JournalBiophys J
PubMed ID12202366
Eukaryotic cells use membrane organelles, like the endoplasmic reticulum or the Golgi, to carry out different functions. Vertebrate rod photoreceptors use hundreds of membrane sacs (the disks) for the detection of light. We have used fluorescent tracers and single cell imaging to study the properties of rod photoreceptor disks. Labeling ... More
Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.
AuthorsEdelson BS, Best TP, Olenyuk B, Nickols NG, Doss RM, Foister S, Heckel A, Dervan PB
JournalNucleic Acids Res
PubMed ID15155849
A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. ... More
Enteropathogenic Escherichia coli translocate Tir and form an intimin-Tir intimate attachment to red blood cell membranes.
AuthorsShaw RK, Daniell S, Frankel G, Knutton S
JournalMicrobiology
PubMed ID11988509
Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC) the type III secreted protein, Tir, is translocated to the host-cell plasma membrane where it functions as a receptor for the bacterial adhesin intimin, leading to intimate bacterial attachment and "attaching and effacing" ... More
Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).
AuthorsGoncharova EA, Goncharov DA, Eszterhas A, Hunter DS, Glassberg MK, Yeung RS, Walker CL, Noonan D, Kwiatkowski DJ, Chou MM, Panettieri RA, Krymskaya VP
JournalJ Biol Chem
PubMed ID12045200
Although the cellular functions of TSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, ... More
The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis.
AuthorsSandgren S, Wittrup A, Cheng F, Jönsson M, Eklund E, Busch S, Belting M
JournalJ Biol Chem
PubMed ID14963039
Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic ... More
Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis.
AuthorsHeinze KG, Koltermann A, Schwille P
JournalProc Natl Acad Sci U S A
PubMed ID10973482
Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different ... More
Molecular cloning and functional expression of the rat 175-kDa hyaluronan receptor for endocytosis.
AuthorsZhou B, Weigel JA, Saxena A, Weigel PH
JournalMol Biol Cell
PubMed ID12181351
We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and approximately 300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were ... More
Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers.
AuthorsMarcinkeviciene J, Kopcho LM, Yang T, Copeland RA, Glass BM, Combs AP, Falahatpisheh N, Thompson L
JournalJ Biol Chem
PubMed ID12029090
Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted ... More
Optimization of spectroscopic and electrophoretic properties of energy transfer primers.
AuthorsHung SC, Mathies RA, Glazer AN
JournalAnal Biochem
PubMed ID9324944
We have synthesized and characterized the spectroscopic properties of 56 energy transfer (ET) fluorescent dye-labeled primers differing in (i) the spacing between the donor and acceptor, (ii) the nature of the spacer (either oligonucleotide or polydideoxyribose phosphate), (iii) the primer sequence (M13 (-40), M13 (-21), M13 reverse, SP6, T3, and ... More
Spatial two-photon fluorescence cross-correlation spectroscopy for controlling molecular transport in microfluidic structures.
AuthorsDittrich PS, Schwille P
JournalAnal Chem
PubMed ID12236358
The increasing availability of microfluidic systems of various geometries and materials for the downscaling of chemical or biochemical processes raises a strong demand for adequate techniques to precisely determine flow parameters and to control fluid and particle manipulation. Of all readout parameters, fluorescence analysis of the fluid or suspended particles ... More
Involvement of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis.
AuthorsNakamura Y, Kawakami M, Yoshihiro A, Miyoshi N, Ohigashi H, Kawai K, Osawa T, Uchida K
JournalJ Biol Chem
PubMed ID11751909
In the present study, we studied the molecular mechanism underlying cell death induced by a cancer chemoprotective compound benzyl isothiocyanate (BITC). The cytotoxic effect of BITC was examined in rat liver epithelial RL34 cells. Apoptosis was induced when the cells were treated with 20 mum BITC, characterized by the appearance ... More
Analysis of human telomerase activity and function by two color single molecule coincidence fluorescence spectroscopy.
AuthorsRen X, Li H, Clarke RW, Alves DA, Ying L, Klenerman D, Balasubramanian S
JournalJ Am Chem Soc
PubMed ID16608333
Telomerase is a nonclassical DNA polymerase that uses its integral RNA as a template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase is unique and involves a translocation step after the synthesis of each telomeric repeat. To directly measure the enzymatic turnover of substrate and the ... More
A new dimension for the development of fluorescence-based assays in solution: from physical principles of FCS detection to biological applications.
AuthorsFöldes-Papp Z, Demel U, Domej W, Tilz GP
JournalExp Biol Med (Maywood)
PubMed ID11976399
Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on ... More