A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection.
AuthorsQian M, Cai D, Verhey KJ, Tsai B,
JournalPLoS Pathog
PubMed ID19503604
'The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py) binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER) where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We ... More
TRPV1 regulators mediate gentamicin penetration of cultured kidney cells.
AuthorsMyrdal SE, Steyger PS
JournalHear Res
PubMed ID15925202
'Transient receptor potential (TRP) receptors are, typically, calcium-permeant cation channels that transduce environmental stimuli. Both kidney epithelial and inner ear sensory cells express TRPV1, are mechanosensors and accumulate the aminoglycoside antibiotic gentamicin. Recently, we showed that Texas Red-conjugated gentamicin (GTTR) enters kidney cells via an endosome-independent pathway. Here, we used ... More
Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis.
AuthorsMyrdal SE, Johnson KC, Steyger PS
JournalHear Res
PubMed ID15925201
'Understanding the cellular mechanism(s) by which the oto- and nephrotoxic aminoglycoside antibiotics penetrate cells, and the precise intracellular distribution of these molecules, will enable identification of aminoglycoside-sensitive targets, and potential uptake blockers. Clones of two kidney cell lines, OK and MDCK, were treated with the aminoglycoside gentamicin linked to the ... More
Interaction of Mycobacterium avium-containing phagosomes with the antigen presentation pathway.
AuthorsUllrich HJ, Beatty WL, Russell DG
JournalJ Immunol
PubMed ID11086039
'Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. ... 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
Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.
'The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates ... More
Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells.
AuthorsNori A, Jensen KD, Tijerina M, Kopecková P, Kopecek J
JournalBioconjug Chem
PubMed ID12526691
'We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic ... More
Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy.
AuthorsSandoval RM, Kennedy MD, Low PS, Molitoris BA
JournalAm J Physiol Cell Physiol
PubMed ID15102609
'Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen ... 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 '
Isolation and lipid composition of apical and basolateral membranes of colonic segments of guinea Pig.
AuthorsMeyer zu Düttingdorf H, Sallmann H, Glockenthör U, von Engelhardt W, Busche R
JournalAnal Biochem
PubMed ID10094774
'In adapting several methods of membrane isolation we established a successful way to purify apical and basolateral membranes of guinea pig colon in a parallel procedure. The conventional purification control by marker enzymes was applied. In addition, luminal membrane proteins were stained with Texas Red. Apical and basolateral enterocyte membranes ... More
High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast.
AuthorsAjo-Franklin CM, Kam L, Boxer SG
JournalProc Natl Acad Sci U S A
PubMed ID11717428
'Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle ... More
A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA.
AuthorsDodge A, Turcatti G, Lawrence I, de Rooij NF, Verpoorte E
JournalAnal Chem
PubMed ID15018583
'This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the ... More
Visual detection of specific, native interactions between soluble and microbead-tethered alpha-helices from membrane proteins.
AuthorsAshish Wimley WC
JournalBiochemistry
PubMed ID11705363
'Using peptides tethered to polymer microbeads, we have developed a technique for measuring the interactions between the transmembrane alpha-helices of membrane proteins and for screening combinatorial libraries of peptides for members that interact with specific helices from membrane proteins. The method was developed using the well-characterized homodimerization sequence of the ... More
Chemistry of sulforhodamine--amine conjugates.
AuthorsCorrie JE, Davis CT, Eccleston JF
JournalBioconjug Chem
PubMed ID11312679
Commercially-available sulforhodamine sulfonyl chlorides contain two isomeric monosulfonyl chlorides. Conjugates of these isomers with amines have different properties because the sulfonamide formed from one isomer can undergo ring-closure to a colorless sultam. This chemistry has been examined for a model conjugate with methylamine and for a bioconjugate with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP. The ... More
Fluorescent histochemical techniques for analysis of intracellular signaling.
AuthorsOksvold MP, Skarpen E, Widerberg J, Huitfeldt HS
JournalJ Histochem Cytochem
PubMed ID11850432
Intracellular signaling relies on the orchestrated cooperation of signaling proteins and modules, their intracellular localization, and membrane trafficking. Recently, a repertoire of fluorescence-based techniques, which significantly increases our potential for detailed studies of the involved mechanisms, has been introduced. Microscopic techniques with increased resolution have been combined with improved techniques ... More
In vivo targeting of organic calcium sensors via genetically selected peptides.
AuthorsMarks KM, Rosinov M, Nolan GP
JournalChem Biol
PubMed ID15123264
A library of constrained peptides that form stable folded structures was screened for aptamers that bind with high affinity to the fluorescent dye Texas red. Two selected clones had binding constants to Texas red of 25 and 80 pM as phage and binding had minimal effects on the fluorescence of ... More
Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling.
AuthorsHenegariu O, Bray-Ward P, Ward DC
JournalNat Biotechnol
PubMed ID10700155
The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for ... More
Assessing the sensitivity of commercially available fluorophores to the intracellular environment.
AuthorsChen AK, Cheng Z, Behlke MA, Tsourkas A,
JournalAnal Chem
PubMed ID18700780
The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute ... More
Interactions of histone H1 with phospholipids and comparison of its binding to giant liposomes and human leukemic T cells.
AuthorsZhao H, Bose S, Tuominen EK, Kinnunen PK,
JournalBiochemistry
PubMed ID15287747
Due to its net positive charge histone H1 readily associates with liposomes containing acidic phospholipids, such as phosphatidylserine (PS). Interestingly, circular dichroism reveals that while histone H1 in aqueous solutions appears as a random coil, its binding to liposomes containing PS is associated with a pronounced increase in alpha-helicity and ... More
Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix.
AuthorsKapoor TM, Mitchison TJ
JournalJ Cell Biol
PubMed ID11564753
We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover ... More
Visualizing the distribution and transport of mRNAs in living cells.
AuthorsBratu DP, Cha BJ, Mhlanga MM, Kramer FR, Tyagi S
JournalProc Natl Acad Sci U S A
PubMed ID14583593
We have visualized the movements of native mRNAs in living cells. Using nuclease-resistant molecular beacons, we imaged the transport and localization of oskar mRNA in Drosophila melanogaster oocytes. When the localization pattern was altered by genetic manipulation of the mRNA's 3' untranslated region, or by chemical perturbation of the intracellular ... More
An ultra-high throughput screening approach for an adenine transferase using fluorescence polarization.
AuthorsLi Z, Mehdi S, Patel I, Kawooya J, Judkins M, Zhang W, Diener K, Lozada A, Dunnington D
JournalJ Biomol Screen
PubMed ID10841598
We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated ... More
Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy.
AuthorsLiu R, Hu D, Tan X, Lu HP
JournalJ Am Chem Soc
PubMed ID16881631
We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in ... More
Synthesis and application of submicrometer fluorescence sensing particles for lysosomal pH measurements in murine macrophages.
AuthorsJi J, Rosenzweig N, Griffin C, Rosenzweig Z
JournalAnal Chem
PubMed ID10952534
Phagocytosis of bioparticles such as bacteria and viruses by macrophages is a critical component of the immune response against infections. In this paper we describe the synthesis of submicrometer fluorescent particles with pH sensing capability. The particles are used to measure the pH and to monitor the effect of chloroquine, ... More
Electric manipulation of bioparticles and macromolecules on microfabricated electrodes.
AuthorsHuang Y, Ewalt KL, Tirado M, Haigis R, Forster A, Ackley D, Heller MJ, O'Connell JP, Krihak M
JournalAnal Chem
PubMed ID11321308
Bioparticle separation, bioparticle enrichment, and electric field-mediated immune detection were carried out on microfabricated semiconductor chips utilizing ac and dc electric fields. Microscale separation on a chip surface having an active area of approximately 16 mm2 was demonstrated for a mixture of Bacillus globigii spores and Escherichia coli bacteria. Dielectrophoretic ... More
Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling.
AuthorsMaamra M, Finidori J, Von Laue S, Simon S, Justice S, Webster J, Dower S, Ross R
JournalJ Biol Chem
PubMed ID10329677
We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, ... More
Helical disposition of proteins and lipopolysaccharide in the outer membrane of Escherichia coli.
AuthorsGhosh AS, Young KD
JournalJ Bacteriol
PubMed ID15743937
In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at ... More
Processing of prothyrotropin-releasing hormone by the family of prohormone convertases.
AuthorsSchaner P, Todd RB, Seidah NG, Nillni EA
JournalJ Biol Chem
PubMed ID9242664
The post-translational processing of prothyrotropin-releasing hormone (pro-TRH25-255) has been extensively studied in our laboratory, and the processing pathway to mature TRH has been elucidated. We have also demonstrated that recombinant PC1 and PC2 process partially purified pro-TRH to cryptic peptides in vitro and that pro-TRH and PC1 mRNAs are coexpressed ... More
Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes.
AuthorsCowan DB, Noria S, Stamm C, Garcia LM, Poutias DN, del Nido PJ, McGowan FX
JournalCirc Res
PubMed ID11249872
We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be internalized to facilitate endotoxin-dependent signal activation in cardiac myocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell line. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized ... More
Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.
AuthorsWang E, Babbey CM, Dunn KW
JournalJ Microsc
PubMed ID15857376
Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system ... More
Bacterial lipopolysaccharide can enter monocytes via two CD14-dependent pathways.
AuthorsKitchens RL, Wang P, Munford RS
JournalJ Immunol
PubMed ID9820530
Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated ... 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
Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.
AuthorsTalaga DS, Lau WL, Roder H, Tang J, Jia Y, DeGrado WF, Hochstrasser RM
JournalProc Natl Acad Sci U S A
PubMed ID11087856
We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. ... More
Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.
AuthorsBuranda T, Lopez GP, Simons P, Pastuszyn A, Sklar LA
JournalAnal Biochem
PubMed ID11700971
Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, ... More
Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo.
AuthorsDai CF, Mangiardi D, Cotanche DA, Steyger PS
JournalHear Res
PubMed ID16466873
Aminoglycoside uptake in the inner ear remains poorly understood. We subcutaneously injected a fluorescently-conjugated aminoglycoside, gentamicin-Texas Red (GTTR), to investigate the in vivo uptake of GTTR in the inner ear of several vertebrates, and in various murine sensory cells using confocal microscopy. In bullfrogs, GTTR uptake was prominent in mature ... More
Texas Res-X and rhodamine Red-X, new derivatives of sulforhodamine 101 and lissamine rhodamine B with improved labeling and fluorescence properties.
AuthorsLefevre C, Kang HC, Haugland RP, Malekzadeh N, Arttamangkul S, Haugland RP
JournalBioconjug Chem
PubMed ID8853462
Texas Red sulfonyl chloride (TR-SC) and Lissamine rhodamine B sulfonyl chloride (LRB-SC) are popular dyes often used to prepare red fluorescent conjugates that are useful second labels in combination with fluorescein. Unfortunately, being sulfonyl chloride derivatives, both are unstable to moisture during storage and prone to hydrolysis in the conjugation ... More