PKC-induced intracellular trafficking of Ca(V)2 precedes its rapid recruitment to the plasma membrane.
AuthorsZhang Y, Helm JS, Senatore A, Spafford JD, Kaczmarek LK, Jonas EA,
JournalJ Neurosci
PubMed ID18322103
'Activation of protein kinase C (PKC) potentiates secretion in Aplysia peptidergic neurons, in part by inducing new sites for peptide release at growth cone terminals. The mechanisms by which ion channels are trafficked to such sites are, however, not well understood. We now show that PKC activation rapidly recruits new ... More
Development of a novel FRET immunosensor technique.
AuthorsLichlyter DJ, Grant SA, Soykan O
JournalBiosens Bioelectron
PubMed ID14611757
'We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab'')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the ... More
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
Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia.
AuthorsDavies JP, Scott C, Oishi K, Liapis A, Ioannou YA
JournalJ Biol Chem
PubMed ID15671032
'NPC1L1, a recently identified relative of Niemann-Pick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 was highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest ... More
Fabrication of phospholipid bilayer-coated microchannels for on-chip immunoassays.
AuthorsYang T, Jung S, Mao H, Cremer PS
JournalAnal Chem
PubMed ID11199961
'Herein we describe a new class of microfluidic immunoassays based upon solid supported lipid bilayers. Two-dimensionally fluid bilayer material, which can accommodate multivalent binding between surface-bound ligands and aqueous receptors, was coated on the surface of poly(dimethylsiloxane) microchannels. The bilayers contained dinitrophenyl (DNP)-conjugated lipids for binding with bivalent anti-DNP antibodies. ... More
Sensitive fluorescence polarization technique for rapid screening of alpha-synuclein oligomerization/fibrillization inhibitors.
AuthorsLuk KC, Hyde EG, Trojanowski JQ, Lee VM,
JournalBiochemistry
PubMed ID17927212
'Parkinson''s disease (PD) is characterized by the accumulation of fibrillar alpha-synuclein (alpha-Syn) inclusions known as Lewy bodies (LBs) and Lewy neurites. Mutations in the alpha-Syn gene or extra copies thereof cause familial PD or dementia with LBs (DLB) in rare kindreds, but abnormal accumulations of wildtype alpha-Syn also are implicated ... More
Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.
AuthorsPanchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY, Haugland RP
JournalJ Histochem Cytochem
PubMed ID10449539
'Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the ... More
Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
'G-protein-coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) ... More
Removal of the membrane-anchoring domain of epidermal growth factor leads to intracrine signaling and disruption of mammary epithelial cell organization.
AuthorsWiley HS, Woolf MF, Opresko LK, Burke PM, Will B, Morgan JR, Lauffenburger DA
JournalJ Cell Biol
PubMed ID9832559
'Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation ... More
Polar localization of virulence-related Esx-1 secretion in mycobacteria.
AuthorsCarlsson F, Joshi SA, Rangell L, Brown EJ,
JournalPLoS Pathog
PubMed ID19180234
'The Esx-1 (type VII) secretion system is critical for virulence of both Mycobacterium tuberculosis and Mycobacterium marinum, and is highly conserved between the two species. Despite its importance, there has been no direct visualization of Esx-1 secretion until now. In M. marinum, we show that secretion of Mh3864, a novel ... More
Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins.
AuthorsStupack DG, Puente XS, Boutsaboualoy S, Storgard CM, Cheresh DA
JournalJ Cell Biol
PubMed ID11684710
'Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the beta subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires ... More
Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging.
AuthorsBestvater F, Spiess E, Stobrawa G, Hacker M, Feurer T, Porwol T, Berchner-Pfannschmidt U, Wotzlaw C, Acker H
JournalJ Microsc
PubMed ID12423261
'Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle ... More
The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.
AuthorsJensen UB, Lowell S, Watt FM
JournalDevelopment
PubMed ID10226000
'In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips ... More
Direct microscopic observation of lettuce leaf decontamination with a prototype fruit and vegetable washing solution and 1% NaCl-NaHCO3.
AuthorsTakeuchi K, Frank JF
JournalJ Food Prot
PubMed ID11510667
'Efficacy of a prototype, food-grade alkaline surfactant washing solution and 1% NaCl-NaHCO3 (pH 10.0) against Escherichia coli O157:H7 cells on lettuce leaves was evaluated. Lettuce was inoculated with 10(9) CFU/ml of E. coli O157:H7 for 24 +/- 1 h at 4 degrees C. Samples were rinsed and treated with the ... More
Photolithographic patterning of polyethylene glycol hydrogels.
AuthorsHahn MS, Taite LJ, Moon JJ, Rowland MC, Ruffino KA, West JL
JournalBiomaterials
PubMed ID16375965
'A simple, inexpensive photolithographic method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithographic masks were prepared by printing the desired patterns onto transparencies using a laser jet printer. Precursor solutions containing monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The ... More
Analysis of proteins stained by Alexa dyes.
AuthorsHuang S, Wang H, Carroll CA, Hayes SJ, Weintraub ST, Serwer P
JournalElectrophoresis
PubMed ID15004835
'Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis ... More
Fluorescent PNA probes as hybridization labels for biological RNA.
AuthorsRobertson KL, Yu L, Armitage BA, Lopez AJ, Peteanu LA
JournalBiochemistry
PubMed ID16681379
'Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts. Noncovalent strategies for labeling RNA rely on attaching fluorescent dyes to hybridization probes which ... More
Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.
'Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of ... 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
Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D.
'The present study describes a novel phenomenon in pancreatic acinar cells undergoing regulated exocytosis. When acinar cell preparations were challenged with the secretagogue carbamylcholine, a subpopulation of zymogen granules became coated with filamentous actin. These zymogen granules were always in proximity of the acinar cell apical membrane (the site of ... More
Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.
AuthorsBacia K, Schwille P,
JournalNat Protoc
PubMed ID18007619
'Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS ... More
Serodiagnosis of infectious diseases with antigen microarrays.
AuthorsBacarese-Hamilton T, Mezzasoma L, Ardizzoni A, Bistoni F, Crisanti A
JournalJ Appl Microbiol
PubMed ID14678154
'AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. ... More
A compact STED microscope providing 3D nanoscale resolution.
AuthorsWildanger D, Medda R, Kastrup L, Hell SW,
JournalJ Microsc
PubMed ID19772534
'The advent of supercontinuum laser sources has enabled the implementation of compact and tunable stimulated emission depletion fluorescence microscopes for imaging far below the diffraction barrier. Here we report on an enhanced version of this approach displaying an all-physics based resolution down to (19 +/- 3) nm in the focal ... More
Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor.
AuthorsWhitson KB, Beechem JM, Beth AH, Staros JV
JournalAnal Biochem
PubMed ID14690686
'We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra ... More
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.
AuthorsHowarth M, Ting AY,
JournalNat Protoc
PubMed ID18323822
'This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and ... More
Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy.
AuthorsSchuler B, Lipman EA, Eaton WA
JournalNature
PubMed ID12384704
'Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) ... More
11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.
AuthorsDe Rosa SC, Herzenberg LA, Herzenberg LA, Roederer M
JournalNat Med
PubMed ID11175858
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
A novel FRET approach for in situ investigation of cellulase-cellulose interaction.
AuthorsWang L, Wang Y, Ragauskas AJ,
JournalAnal Bioanal Chem
PubMed ID20694721
A novel real-time in situ detection method for the investigation of cellulase-cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling ... More
A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair.
AuthorsBritton S, Coates J, Jackson SP,
Journal
PubMed ID23897892
DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair ... More
Synthesis, characterization, and biological evaluation of integrin alphavbeta3-targeted PAMAM dendrimers.
AuthorsBoswell CA, Eck PK, Regino CA, Bernardo M, Wong KJ, Milenic DE, Choyke PL, Brechbiel MW,
JournalMol Pharm
PubMed ID18537262
Ligand size and valency strongly influence the receptor uptake and clearance of tumor angiogenesis imaging agents. The structures of successful imaging agents exhibit a high degree of variability, encompassing small monovalent arginine-glycine-aspartic acid (RGD)-containing peptides, multivalent RGD-oligomers, and a monoclonal antibody against integrin alpha-v-beta-3 (alpha-v-beta-3). We have pursued a nanoscale ... More
Effect of flexibility and cis residues in single-molecule FRET studies of polyproline.
AuthorsBest RB, Merchant KA, Gopich IV, Schuler B, Bax A, Eaton WA,
JournalProc Natl Acad Sci U S A
PubMed ID18029448
Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Förster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to ... More
Pericellular hyaluronan coat visualized in live cells with a fluorescent probe is scaffolded by plasma membrane protrusions.
AuthorsRilla K, Tiihonen R, Kultti A, Tammi M, Tammi R,
JournalJ Histochem Cytochem
PubMed ID18574248
Many cell types wear up to 20-mum-wide hyaluronidase-sensitive surface coats, detected by exclusion of sedimenting particles like fixed erythrocytes. The structure of the coat is enigmatic, being apparently too thick to be accounted by random coils or even extended chains of just hyaluronan attached to cell surface. We have shown ... More
DNA methylation promotes Aurora-B-driven phosphorylation of histone H3 in chromosomal subdomains.
AuthorsMonier K, Mouradian S, Sullivan KF,
JournalJ Cell Sci
PubMed ID17164288
Confinement of enzymatic reactions to nuclear and chromosomal subdomains regulates functional organization of the nucleus. Aurora-B kinase regulates cell-cycle-dependent phosphorylation of chromosomal substrates through sequential localization to a series of sites on chromosomes and the mitotic spindle. In G2 nuclei, Aurora-B recruitment to heterochromatin restricts histone H3S10 phosphorylation to a ... More
Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth.
AuthorsPatel N, Singh SB, Basu SK, Mukhopadhyay A,
JournalProc Natl Acad Sci U S A
PubMed ID18319337
Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog ... More
Real-time spectrofluorometric assays for the lumenal environment of the maturing phagosome.
AuthorsYates RM, Russell DG,
JournalMethods Mol Biol
PubMed ID18425459
The ultimate goal of phagosomal maturation is the delivery of internalized, particulate cargo to acidic, hydrolytically competent compartments capable of mediating its degradation. Here we outline in detail three fluorometric techniques that allow the study of phagosomal maturation in macrophages by quantifying functionally important features of the lumenal environment of ... More
Vascular targeted nanoparticles for imaging and treatment of brain tumors.
AuthorsReddy GR, Bhojani MS, McConville P, Moody J, Moffat BA, Hall DE, Kim G, Koo YE, Woolliscroft MJ, Sugai JV, Johnson TD, Philbert MA, Kopelman R, Rehemtulla A, Ross BD,
JournalClin Cancer Res
PubMed ID17121886
PURPOSE: Development of new therapeutic drug delivery systems is an area of significant research interest. The ability to directly target a therapeutic agent to a tumor site would minimize systemic drug exposure, thus providing the potential for increasing the therapeutic index. EXPERIMENTAL DESIGN: Photodynamic therapy (PDT) involves the uptake of ... More
Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.
AuthorsCox WG, Singer VL,
JournalBiotechniques
PubMed ID14740493
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized ... More
Programmable in situ amplification for multiplexed imaging of mRNA expression.
AuthorsChoi HM, Chang JY, Trinh le A, Padilla JE, Fraser SE, Pierce NA,
JournalNat Biotechnol
PubMed ID21037591
In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. ... More
Competitive binding assay for glucose based on glycodendrimer-fluorophore conjugates.
A new fluorescent glucose assay has been created using Alexa Fluor 647-labeled concanavalin A (Con A) and a fourth-generation PAMAM Alexa Fluor 594-labeled glycodendrimer. This assay has been shown to have a large response to glucose within the biological range and to be capable of functioning within a polymer hydrogel. ... More
MACROH2A2, a new member of the MARCOH2A core histone family.
AuthorsCostanzi C, Pehrson JR
JournalJ Biol Chem
PubMed ID11262398
MACROH2As are core histones that have a unique hybrid structure consisting of an amino-terminal domain that closely resembles a full-length histone H2A followed by a large nonhistone region. The human MACROH2A1 gene, on chromosome 5, encodes two MACROH2A subtypes, MACROH2A1.1 and MACROH2A1.2, produced by alternate splicing. Here we report the ... More
Three-dimensional spectral imaging by hadamard transform spectroscopy in a programmable array microscope.
We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene ... More
Simultaneous imaging and functional assessment of cytoskeletal protein connections in passively loaded single muscle cells.
AuthorsShah SB, Lieber RL
JournalJ Histochem Cytochem
PubMed ID12502751
We describe a novel system that permits simultaneous confocal imaging of protein interactions and measurement of cell mechanical properties during passive loading. A mechanical apparatus was designed to replace the stage of a confocal microscope, enabling cell manipulation, force transduction, and imaging. In addition, image processing algorithms were developed to ... More
Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin.
The mechanism of action of Endostatin, an endogenous inhibitor of angiogenesis and tumor growth, remains unknown. We utilized phage-display technology to identify polypeptides that mimic the binding domains of proteins with which Endostatin interacts. A conformed peptide (E37) was identified that shares an epitope with human tropomyosin implicating tropomyosin as ... More
ErbB-2 amplification inhibits down-regulation and induces constitutive activation of both ErbB-2 and epidermal growth factor receptors.
AuthorsWorthylake R, Opresko LK, Wiley HS
JournalJ Biol Chem
PubMed ID10085130
ErbB-2/HER2 is an important signaling partner for the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we used a human mammary epithelial cell system in which erbB-2 expression was increased ... More
PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization.
AuthorsSun Y, Carroll S, Kaksonen M, Toshima JY, Drubin DG
JournalJ Cell Biol
PubMed ID17452534
The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P(2)) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels ... More
Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence.
AuthorsSchuler B, Lipman EA, Steinbach PJ, Kumke M, Eaton WA
JournalProc Natl Acad Sci U S A
PubMed ID15699337
To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those ... More
Analysis of DNA-dependent protein kinase-mediated DNA end joining by two-photon fluorescence cross-correlation spectroscopy.
AuthorsMerkle D, Block WD, Yu Y, Lees-Miller SP, Cramb DT
JournalBiochemistry
PubMed ID16566590
Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of ... More
Real-time imaging of fluorescent flagellar filaments.
AuthorsTurner L, Ryu WS, Berg HC
JournalJ Bacteriol
PubMed ID10781548
Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments ... More
The development, maturation, and turnover rate of mouse spleen dendritic cell populations.
AuthorsKamath AT, Pooley J, O'Keeffe MA, Vremec D, Zhan Y, Lew AM, D'Amico A, Wu L, Tough DF, Shortman K
JournalJ Immunol
PubMed ID11120796
Three distinct subtypes of dendritic cells (DC) are present in mouse spleen, separable as CD4(-)8alpha(-), CD4(+)8alpha(-), and CD4(-)8alpha(+) DC. We have tested whether these represent stages of development or activation within one DC lineage, or whether they represent separate DC lineages. All three DC subtypes appear relatively mature by many ... More
Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.
AuthorsFukami K, Yoshida M, Inoue T, Kurokawa M, Fissore RA, Yoshida N, Mikoshiba K, Takenawa T
JournalJ Cell Biol
PubMed ID12695499
Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we ... More
Mechanism of uptake of C105Y, a novel cell-penetrating peptide.
AuthorsRhee M, Davis P
JournalJ Biol Chem
PubMed ID16272160
C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359-374 of alpha1-antitrypsin, enhances gene expression from DNA nanoparticles. To investigate how this enhancement occurs, C105Y was fluorescently labeled to study its uptake and intracellular trafficking. When human hepatoma cells (HuH7) were incubated with fluorescently labeled ... More
B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection.
AuthorsBaumgarth N, Herman OC, Jager GC, Brown LE, Herzenberg LA, Chen J
JournalJ Exp Med
PubMed ID10899913
We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell-derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus ... More
Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition.
Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels ... More
Identification of a germ-line pro-B cell subset that distinguishes the fetal/neonatal from the adult B cell development pathway.
AuthorsLu LS, Tung J, Baumgarth N, Herman O, Gleimer M, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID11867763
Studies presented here show that the expression of CD4, MHC class II (Ia,) and B220 cleanly resolves a major and a minor subset within the earliest pro-B cell population (germ-line pro-B) in adult bone marrow (BM). The major subset expresses intermediate B220 and low CD4 levels. The minor subset, which ... 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
Acetylcholinesterase dynamics at the neuromuscular junction of live animals.
AuthorsKrejci E, Martinez-Pena y Valenzuela I, Ameziane R, Akaaboune M
JournalJ Biol Chem
PubMed ID16455662
At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely ... More
Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes.
We studied fluorescence intensity, polarization and lifetime of some commonly used fluorophores conjugated to oligodeoxyribonucleotides with different primary and secondary structures. We found that fluorescence intensity can increase or decrease upon hybridization of the labeled strand to its complement depending on the sequence and position of the fluorophore. Up to ... More
Selective cell uptake of modified Tat peptide-fluorophore conjugates in rat retina in ex vivo and in vivo models.
AuthorsBarnett EM, Elangovan B, Bullok KE, Piwnica-Worms D
JournalInvest Ophthalmol Vis Sci
PubMed ID16723475
PURPOSE: To determine the pattern of retinal uptake of modified Tat peptide-fluorophore conjugates in the rat after ex vivo application and intravitreal injection. METHODS: Modified Tat peptide (RKKRRORRRGC) was conjugated at the C terminus to Alexa Fluor 594 to enable visualization of uptake. In the ex vivo model, posterior segments ... More
Quantitative determination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection.
AuthorsTakeuchi K, Frank JF
JournalJ Food Prot
PubMed ID11271758
Viability of Escherichia coli O157:H7 cells on lettuce leaves after 200 mg/liter (200 ppm) chlorine treatment and the role of lettuce leaf structures in protecting cells from chlorine inactivation were evaluated by confocal scanning microscopy (CSLM). Lettuce samples (2 by 2 cm) were inoculated by immersing in a suspension containing ... More
Translocation of PKC[theta] in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C.
AuthorsVillalba M, Bi K, Hu J, Altman Y, Bushway P, Reits E, Neefjes J, Baier G, Abraham RT, Altman A
JournalJ Cell Biol
PubMed ID11956228
PKCtheta plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-kappaB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCtheta to ... More
Triple-target microarray experiments: a novel experimental strategy.
AuthorsForster T, Costa Y, Roy D, Cooke HJ, Maratou K
JournalBMC Genomics
PubMed ID15018645
BACKGROUND: High-throughput, parallel gene expression analysis by means of microarray technology has become a widely used technique in recent years. There are currently two main dye-labelling strategies for microarray studies based on custom-spotted cDNA or oligonucleotides arrays: (I) Dye-labelling of a single target sample with a particular dye, followed by ... More
Lipid microdomain clustering induces a redistribution of antigen recognition and adhesion molecules on human T lymphocytes.
AuthorsMitchell JS, Kanca O, McIntyre BW
JournalJ Immunol
PubMed ID11884440
The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with ... More
Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy.
AuthorsRen Y, Ridsdale A, Coderre E, Stys PK
JournalJ Neurosci Methods
PubMed ID11040413
Intracellular Ca(2+) plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca(2+) is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca(2+) dynamics in neurons. In constrast, there is virtually no such information for ... More
Synthetic mimics of small Mammalian cell surface receptors.
AuthorsBoonyarattanakalin S, Martin SE, Dykstra SA, Peterson BR
JournalJ Am Chem Soc
PubMed ID15600339
Receptors on the surface of mammalian cells promote the uptake of cell-impermeable ligands by receptor-mediated endocytosis. To mimic this process, we synthesized small molecules designed to project anti-dinitrophenyl antibody-binding motifs from the surface of living Jurkat lymphocytes. These synthetic receptors comprise N-alkyl derivatives of 3beta-cholesterylamine as the plasma membrane anchor ... More
Multiplex real-time single nucleotide polymorphism detection and quantification by quencher extension.
AuthorsRudi K, Zimonja M, Hannevik SE, Drømtorp SM
JournalBiotechniques
PubMed ID16568821
Multiplex quencher extension (multiplex-QEXT) is a novel closed tube single-step method for detection and quantification of several single nucleotide polymorphisms (SNPs) simultaneously. The principle of multiplex-QEXT is that 5' reporter-labeled probes are 3' single-base-extended with TAMRA dideoxy nucleotides if the respective SNP alleles are present. TAMRA can serve as either ... More
Screening for aneuploidies of ten different chromosomes in two rounds of FISH: a short and reliable protocol.
AuthorsBaart EB, Martini E, Van Opstal D
JournalPrenat Diagn
PubMed ID15614916
OBJECTIVE: To develop a DNA labelling protocol for the simultaneous detection of five different fluorescent chromosomal DNA probes within one round of hybridisation. In combination with a commercial five-colour probemix for the second round of hybridisation, this results in a fast and reliable Fluorescence in situ Hybridization (FISH) protocol, enabling ... More
Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry.
AuthorsZahavy E, Fisher M, Bromberg A, Olshevsky U
JournalAppl Environ Microbiol
PubMed ID12676717
Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET ... More
Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation.
AuthorsGhosh A, Mazarakos K, Zhou HX
JournalProc Natl Acad Sci U S A
PubMed ID31506351
Membraneless organelles, corresponding to the droplet phase upon liquid-liquid phase separation (LLPS) of protein or protein-RNA mixtures, mediate myriad cellular functions. Cells use a variety of biochemical signals such as expression level and posttranslational modification to regulate droplet formation and dissolution, but the physical basis of the regulatory mechanisms remains ... More
CSD-Induced Arterial Dilatation and Plasma Protein Extravasation Are Unaffected by Fremanezumab: Implications for CGRP's Role in Migraine with Aura.
AuthorsSchain AJ, Melo-Carrillo A, Stratton J, Strassman AM, Burstein R
JournalJ Neurosci
PubMed ID31127003
Cortical spreading depression (CSD) is a wave of neuronal depolarization thought to underlie migraine aura. Calcitonin gene-related peptide (CGRP) is a potent vasodilator involved in migraine pathophysiology. Evidence for functional connectivity between CSD and CGRP has triggered scientific interest in the possibility that CGRP antagonism may disrupt vascular responses to ... More
Hyaluronic Acid Hydrogel Integrated with Mesenchymal Stem Cell-Secretome to Treat Endometrial Injury in a Rat Model of Asherman's Syndrome.
AuthorsLiu F, Hu S, Yang H, Li Z, Huang K, Su T, Wang S, Cheng K
JournalAdv Healthc Mater
PubMed ID31148407
Stem cell therapies have made strides toward the efficacious treatment of injured endometrium and the prevention of intrauterine adhesions, or Asherman's syndrome (AS). Despite this progress, they are limited by their risk of tumor formation, low engraftment rates, as well as storage and transportation logistics. While attempts have been made ... More
High macrophage activities are associated with advanced periductal fibrosis in chronic Opisthorchis viverrini infection.
AuthorsSalao K, Watakulsin K, Mairiang E, Suttiprapa S, Tangkawattana S, Edwards SW, Sripa B
JournalParasite Immunol
PubMed ID30449026
Liver fluke infection caused by Opisthorchis viverrini induces several hepatobiliary conditions including advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), but >25% of the infected population develops APF and 1% develop CCA. The innate immune response is the first line of defence, and macrophages are critical regulators of fibrosis. We hypothesized ... More
Mapping Neurotransmitter Identity in the Whole-Mount
AuthorsMeissner GW, Nern A, Singer RH, Wong AM, Malkesman O, Long X
JournalGenetics
PubMed ID30563859
Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence ... More
LrrkA, a kinase with leucine-rich repeats, links folate sensing with Kil2 activity and intracellular killing.
AuthorsBodinier R, Leiba J, Sabra A, Jauslin TN, Lamrabet O, Guilhen C, Marchetti A, Iwade Y, Kawata T, Lima WC, Cosson P
JournalCell Microbiol
PubMed ID31652367
Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested ... More