'Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold ... More
Glycosylation influences the lectin activities of the macrophage mannose receptor.
AuthorsSu Y, Bakker T, Harris J, Tsang C, Brown GD, Wormald MR, Gordon S, Dwek RA, Rudd PM, Martinez-Pomares L
JournalJ Biol Chem
PubMed ID15983039
'The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. ... More
An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.
AuthorsGhosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR
JournalJ Cell Biol
PubMed ID9722606
'To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38''s postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it ... 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
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
Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules.
AuthorsStraley KS, Green SA
JournalJ Cell Biol
PubMed ID11018057
'Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved ... More
Antibody interactions with the capsule of Cryptococcus neoformans.
AuthorsFeldmesser M, Rivera J, Kress Y, Kozel TR, Casadevall A
JournalInfect Immun
PubMed ID10816523
'Monoclonal antibodies to the encapsulated fungus Cryptococcus neoformans produce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF ... 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
Multiplex detection of surface molecules on colorectal cancers.
AuthorsEllmark P, Belov L, Huang P, Lee CS, Solomon MJ, Morgan DK, Christopherson RI
JournalProteomics
PubMed ID16485257
'A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was ... More
Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake.
AuthorsCai L, de Beer MC, de Beer FC, van der Westhuyzen DR
JournalJ Biol Chem
PubMed ID15561721
'Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated ... More
Physical interactions of the peroxisomal targeting signal 1 receptor pex5p, studied by fluorescence correlation spectroscopy.
AuthorsWang D, Visser NV, Veenhuis M, van der Klei IJ
JournalJ Biol Chem
PubMed ID12930827
'We have studied Hansenula polymorpha Pex5p and Pex8p using fluorescence correlation spectroscopy (FCS). Pex5p is the Peroxisomal Targeting Signal 1 (PTS1) receptor and Pex8p is an intraperoxisomal protein. Both proteins are essential for PTS1 protein import and have been shown to physically interact. We used FCS to analyze the molecular ... More
Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells.
AuthorsLarson DR, Gosse JA, Holowka DA, Baird BA, Webb WW
JournalJ Cell Biol
PubMed ID16275755
'Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also ... More
Evidence for activation of tissue factor by an allosteric disulfide bond.
'Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF ... More
Actin and phosphoinositide binding by the ActA protein of the bacterial pathogen Listeria monocytogenes.
AuthorsCicchetti G, Maurer P, Wagener P, Kocks C
JournalJ Biol Chem
PubMed ID10559250
'The surface protein ActA of the pathogenic bacterium Listeria monocytogenes induces actin-driven movement of bacteria in the cytoplasm of infected host cells and serves as a model for actin-based motility in general. We generated and purified soluble recombinant fragments of ActA and assessed their ability to interact with the acidic ... More
A fluorescence assay for peptide translocation into mitochondria.
AuthorsMartinez-Caballero S, Peixoto PM, Kinnally KW, Campo ML
JournalAnal Biochem
PubMed ID17240346
'Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with ... More
Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters.
AuthorsDaniels GM, Amara SG
JournalJ Biol Chem
PubMed ID10585462
'The dopamine transporter plays an essential role in the modulation of dopaminergic neurotransmission by mediating the reuptake of dopamine into presynaptic neurons. In cells expressing the dopamine transporter, activation of protein kinase C by phorbol esters results in a significant reduction in dopamine uptake. This phorbol ester-mediated inhibition of dopamine ... 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
Remodeling of the bacterial RNA polymerase supramolecular complex in response to environmental conditions.
AuthorsVerma S, Xiong Y, Mayer MU, Squier TC
JournalBiochemistry
PubMed ID17319694
'Directed binding of RNA polymerase to distinct promoter elements controls transcription and promotes adaptive responses to changing environmental conditions. To identify proteins that modulate transcription, we have expressed a tagged alpha-subunit of RNA polymerase in Shewanella oneidensis under controlled growth conditions, isolated the protein complex using newly developed multiuse affinity ... More
Dermal microvascular endothelial cells express the 180-kDa macrophage mannose receptor in situ and in vitro.
AuthorsGröger M, Holnthoner W, Maurer D, Lechleitner S, Wolff K, Mayr BB, Lubitz W, Petzelbauer P
JournalJ Immunol
PubMed ID11067894
'Expression of the 180-kDa mannose receptor (MR) is mainly found on cells of the macrophage lineage. MR mediates the uptake of micro-organisms and host-derived glycoproteins. We demonstrate that endothelium of the human skin in situ and dermal microvascular endothelial cells (DMEC) in vitro expressed MR at both the protein and ... More
Trajectory of nucleosomal linker DNA studied by fluorescence resonance energy transfer.
AuthorsTóth K, Brun N, Langowski J
JournalBiochemistry
PubMed ID11389607
'While the structure of the nucleosome core is known in atomic detail, the precise geometry of the DNA beyond the core particle is still unknown. We have used fluorescence resonance energy transfer (FRET) for determining the end-to-end distance of DNA fragments assembled with histones into nucleosomes. The DNA of a ... More
Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin.
AuthorsCaron NS, Desmond CR, Xia J, Truant R,
Journal
PubMed ID23898200
'Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, ... More
Channel formation by the glycosylphosphatidylinositol-anchored protein binding toxin aerolysin is not promoted by lipid rafts.
AuthorsNelson KL, Buckley JT
JournalJ Biol Chem
PubMed ID10770947
'Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment ... More
Bacterial DNA segregation by dynamic SopA polymers.
AuthorsLim GE, Derman AI, Pogliano J
JournalProc Natl Acad Sci U S A
PubMed ID16306264
'Many bacterial plasmids and chromosomes rely on ParA ATPases for proper positioning within the cell and for efficient segregation to daughter cells. Here we demonstrate that the F-plasmid-partitioning protein SopA polymerizes into filaments in an ATP-dependent manner in vitro, and that the filaments elongate at a rate that is similar ... More
Zero mode waveguides for single-molecule spectroscopy on lipid membranes.
'Zero mode waveguides (ZMWs), subwavelength optical nanostructures with dimensions ranging from 50 to 200 nm, have been used to study systems involving ligand-receptor interactions. We show that under proper conditions, lipid membranes will invaginate into the nanostructures, which confine optical excitation to subattoliter volumes. Fluorescence correlation spectroscopy (FCS) was used ... More
Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips.
'MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 ... 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
Rapid isolation of high-affinity protein binding peptides using bacterial display.
AuthorsBessette PH, Rice JJ, Daugherty PS
JournalProtein Eng Des Sel
PubMed ID15531628
'A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface ... More
Robust isolation of malignant plasma cells in multiple myeloma.
AuthorsFrigyesi I, Adolfsson J, Ali M, Kronborg Christophersen M, Johnsson E, Turesson I, Gullberg U, Hansson M, Nilsson B,
Journal
PubMed ID24385542
'Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification in multiple myeloma. However, the malignant plasma cells represent a relatively small subset of bone marrow cells, and need to be enriched prior to analysis. Currently, the cell surface marker CD138 (SDC1) is used for this ... More
Heterogeneity in neutrophil microparticles reveals distinct proteome and functional properties.
AuthorsDalli J, Montero-Melendez T, Norling LV, Yin X, Hinds C, Haskard D, Mayr M, Perretti M,
Journal
PubMed ID23660474
'Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension ... More
Printing proteins as microarrays for high-throughput function determination.
AuthorsMacBeath G, Schreiber SL
JournalScience
PubMed ID10976071
'Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A ... More
Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.
AuthorsGarcia-Rodriguez C, Levy R, Arndt JW, Forsyth CM, Razai A, Lou J, Geren I, Stevens RC, Marks JD
JournalNat Biotechnol
PubMed ID17173035
'Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with ... More
Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils.
AuthorsSchagat TL, Wofford JA, Wright JR
JournalJ Immunol
PubMed ID11160338
'Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils ... More
Carbon nanotubes as intracellular protein transporters: generality and biological functionality.
AuthorsKam NW, Dai H
JournalJ Am Chem Soc
PubMed ID15839702
'Various proteins adsorb spontaneously on the sidewalls of acid-oxidized single-walled carbon nanotubes. This simple nonspecific binding scheme can be used to afford noncovalent protein-nanotube conjugates. The proteins are found to be readily transported inside various mammalian cells with nanotubes acting as the transporter via the endocytosis pathway. Once released from ... 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
Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells.
AuthorsJames JR, White SS, Clarke RW, Johansen AM, Dunne PD, Sleep DL, Fitzgerald WJ, Davis SJ, Klenerman D,
JournalProc Natl Acad Sci U S A
PubMed ID17971442
'The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster of differentiation (CD) 3epsilondelta, epsilongamma, and zetazeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alphabeta heterodimer per complex. ... More
Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells.
AuthorsOkuyama M, Laman H, Kingsbury SR, Visintin C, Leo E, Eward KL, Stoeber K, Boshoff C, Williams GH, Selwood DL
JournalNat Methods
PubMed ID17220893
'We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an ... More
Fluorescence linked immunosorbant assays using microtiter plates.
AuthorsVelappan N, Clements J, Kiss C, Valero-Aracama R, Pavlik P, Bradbury AR,
JournalJ Immunol Methods
PubMed ID18514691
Fluorescence methods are widely used in the detection of antibodies and other binding events. However, as a general screening and detection tool in microtiter plates, enzyme linked immunosorbant (ELISA) methods predominate. In this paper we explore all parameters for effective use of fluorescence as a plate based detection method, including ... More
CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis.
Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that 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
Requirement for RORgamma in thymocyte survival and lymphoid organ development.
AuthorsSun Z, Unutmaz D, Zou YR, Sunshine MJ, Pierani A, Brenner-Morton S, Mebius RE, Littman DR
JournalScience
PubMed ID10875923
Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor RORgamma lose thymic expression of the anti-apoptotic factor Bcl-xL. RORgamma thus regulates the survival of CD4+8+ thymocytes and may control the ... More
OmpA targets dendritic cells, induces their maturation and delivers antigen into the MHC class I presentation pathway.
AuthorsJeannin P, Renno T, Goetsch L, Miconnet I, Aubry JP, Delneste Y, Herbault N, Baussant T, Magistrelli G, Soulas C, Romero P, Cerottini JC, Bonnefoy JY
JournalNat Immunol
PubMed ID11101872
We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin ... More
Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.
AuthorsFalati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B
JournalNat Med
PubMed ID12244306
We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. ... More
Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation.
AuthorsPhan TG, Green JA, Gray EE, Xu Y, Cyster JG,
JournalNat Immunol
PubMed ID19503106
Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on ... More
Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons.
AuthorsTerada S, Kinjo M, Hirokawa N
JournalCell
PubMed ID11051554
Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser ... 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
The imprinted NPAP1/C15orf2 gene in the Prader-Willi syndrome region encodes a nuclear pore complex associated protein.
AuthorsNeumann LC, Markaki Y, Mladenov E, Hoffmann D, Buiting K, Horsthemke B,
JournalHum Mol Genet
PubMed ID22694955
The Prader-Willi syndrome (PWS) region in 15q11q13 harbours a cluster of imprinted genes expressed from the paternal chromosome only. Whereas loss of function of the SNORD116 genes appears to be responsible for the major features of PWS, the role of the other genes is less clear. One of these genes ... More
3D-structured illumination microscopy provides novel insight into architecture of human centrosomes.
Centrioles are essential for the formation of cilia and flagella. They also form the core of the centrosome, which organizes microtubule arrays important for cell shape, polarity, motility and division. Here, we have used super-resolution 3D-structured illumination microscopy to analyse the spatial relationship of 18 centriole and pericentriolar matrix (PCM) ... More
Avoiding nonspecific interactions in studies of the plasma proteome: practical solutions to prevention of nonspecific interactions for label-free detection of low-abundance plasma proteins.
The molecular constitution of blood can be highly representative of the physiological state of an individual and offers an ideal target for studies of biomarkers. High-abundance plasma proteins, particularly albumin, dominate the plasma proteome, but it is the low-abundance proteins (such as cytokines) that are commonly associated with many pathophysiological ... More
Spatial distribution of DARPP-32 in dendritic spines.
AuthorsBlom H, Rönnlund D, Scott L, Westin L, Widengren J, Aperia A, Brismar H,
Journal
PubMed ID24058659
The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3´, 5´-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated ... More
Stoichiometry of the murine gammadelta T cell receptor.
AuthorsHayes SM, Love PE,
JournalJ Exp Med
PubMed ID16418397
The T cell receptor for antigen (TCR) complex is organized into two functional domains: the antigen-binding clonotypic heterodimer and the signal-transducing invariant CD3 and TCRzeta chains. In most vertebrates, there are two different clonotypic heterodimers (TCRalphabeta and TCRgammadelta) that define the alphabeta and gammadelta T cell lineages, respectively. alphabeta- and ... More
Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.
AuthorsBíró A, Cervenak L, Balogh A, Lorincz A, Uray K, Horváth A, Romics L, Matkó J, Füst G, László G,
JournalJ Lipid Res
PubMed ID17023738
Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely ... More
A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them.
AuthorsCarman CV, Springer TA
JournalJ Cell Biol
PubMed ID15504916
The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure. This endothelial ... More
A small molecule agonist of an integrin, alphaLbeta2.
AuthorsYang W, Carman CV, Kim M, Salas A, Shimaoka M, Springer TA
JournalJ Biol Chem
PubMed ID17023419
The binding of integrin alpha(L)beta(2) to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of alpha(L)beta(2) are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described alpha/beta I allosteric inhibitor, ... More
Outer membrane protein A (OmpA) binds to and activates human macrophages.
AuthorsSoulas C, Baussant T, Aubry JP, Delneste Y, Barillat N, Caron G, Renno T, Bonnefoy JY, Jeannin P
JournalJ Immunol
PubMed ID10946255
Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4 degrees C) to murine and human macrophages in a dose-dependent manner and ... More
Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells.
AuthorsOno T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T
JournalCell
PubMed ID14532007
The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) ... More
Direct isolation of human central nervous system stem cells.
AuthorsUchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL
JournalProc Natl Acad Sci U S A
PubMed ID11121071
Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These ... More
Detection of pyruvate dehydrogenase E1 alpha-subunit deficiencies in females by immunohistochemical demonstration of mosaicism in cultured fibroblasts.
AuthorsLib MY, Brown RM, Brown GK, Marusich MF, Capaldi RA
JournalJ Histochem Cytochem
PubMed ID12070266
Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on ... More
Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.
AuthorsMallet WG, Maxfield FR
JournalJ Cell Biol
PubMed ID10465644
Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins ... More
Tests of the extension and deadbolt models of integrin activation.
AuthorsZhu J, Boylan B, Luo BH, Newman PJ, Springer TA
JournalJ Biol Chem
PubMed ID17301049
Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt ... More
Alpha4 integrins are type I cAMP-dependent protein kinase-anchoring proteins.
AuthorsLim CJ, Han J, Yousefi N, Ma Y, Amieux PS, McKnight GS, Taylor SS, Ginsberg MH
JournalNat Cell Biol
PubMed ID17369818
A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). ... 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
Preformed oligomeric epidermal growth factor receptors undergo an ectodomain structure change during signaling.
AuthorsMartin-Fernandez M, Clarke DT, Tobin MJ, Jones SV, Jones GR
JournalBiophys J
PubMed ID11964230
Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, ... 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
One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes.
AuthorsWallrabe H, Stanley M, Periasamy A, Barroso M
JournalJ Biomed Opt
PubMed ID12880337
One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding ... 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
HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity.
AuthorsKensch O, Connolly BA, Steinhoff HJ, McGregor A, Goody RS, Restle T
JournalJ Biol Chem
PubMed ID10751399
Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted ... More
Uptake of lipoproteins for axonal growth of sympathetic neurons.
AuthorsPosse De Chaves EI, Vance DE, Campenot RB, Kiss RS, Vance JE
JournalJ Biol Chem
PubMed ID10867025
Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high ... More
Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells.
AuthorsIida T, Ohno H, Nakaseko C, Sakuma M, Takeda-Ezaki M, Arase H, Kominami E, Fujisawa T, Saito T
JournalJ Immunol
PubMed ID11046036
CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of ... 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
Unusual electrostatic effects on binding of C1q to anionic liposomes: role of anionic phospholipid domains and their line tension.
AuthorsBradley AJ, Maurer-Spurej E, Brooks DE, Devine DV
JournalBiochemistry
PubMed ID10387057
The binding of 125I-C1q to anionic liposomes was studied as a function of protein concentration, pH, ionic strength, and anionic lipid composition. The maximum amount of protein bound per micromole of lipid was very sensitive to electrostatic factors, increasing strongly with decreased pH and ionic strength or increased anionic lipid ... More
Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins.
AuthorsOsaki T, Omotezako M, Nagayama R, Hirata M, Iwanaga S, Kasahara J, Hattori J, Ito I, Sugiyama H, Kawabata S
JournalJ Biol Chem
PubMed ID10473569
Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin ... More
Matrix metalloproteinase-9 undergoes expression and activation during dendritic remodeling in adult hippocampus.
AuthorsSzklarczyk A, Lapinska J, Rylski M, McKay RD, Kaczmarek L
JournalJ Neurosci
PubMed ID11826121
Neurons of adult brain are able to remodel their synaptic connections in response to various stimuli. Modifications of the peridendritic environment, including the extracellular matrix, are likely to play a role during synapse remodeling. Proteolytic disassembly of ECM is a complex process using the regulated actions of specific extracellular proteinases. ... More
Real-time observations of individual macaque sperm undergoing tight binding and the acrosome reaction on the zona pellucida.
Changes in binding affinity, acrosomal status, and motility of living sperm on the zona pellucida were for the first time in any mammalian species directly observed and analyzed with video microscopy. A single zona was air-dried and rehydrated on a microscope slide, and a coverslip supported by glass beads was ... More
Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors.
Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X(7) receptor (previously termed P2Z). These responses include opening of a cation-selective channel/pore that allows entry of the fluorescent dye ethidium and activation of a membrane metalloprotease that sheds the ... More
The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer's patch high endothelial venules.
AuthorsWarnock RA, Campbell JJ, Dorf ME, Matsuzawa A, McEvoy LM, Butcher EC
JournalJ Exp Med
PubMed ID10620606
Chemokines have been hypothesized to contribute to the selectivity of lymphocyte trafficking not only as chemoattractants, but also by triggering integrin-dependent sticking (arrest) of circulating lymphocytes at venular sites of extravasation. We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments ... More
Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis.
AuthorsPankov R, Cukierman E, Katz BZ, Matsumoto K, Lin DC, Lin S, Hahn C, Yamada KM
JournalJ Cell Biol
PubMed ID10704455
Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates ... More
Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell.
AuthorsZhao T, Newman PJ
JournalJ Cell Biol
PubMed ID11149921
Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, ... More
Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.
AuthorsHowell BJ, Hoffman DB, Fang G, Murray AW, Salmon ED
JournalJ Cell Biol
PubMed ID10995431
The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and ... More
Binding and internalization of lipopolysaccharide by Cla-1, a human orthologue of rodent scavenger receptor B1.
AuthorsVishnyakova TG, Bocharov AV, Baranova IN, Chen Z, Remaley AT, Csako G, Eggerman TL, Patterson AP
JournalJ Biol Chem
PubMed ID12651854
Scavenger receptor, class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester. SR-BI recognizes HDL, low density lipoprotein (LDL), exchangeable apolipoproteins, and protein-free lipid vesicles containing negatively charged phospholipids. Lipopolysaccharides (LPS) are highly glycosylated anionic phospholipids contributing to septic shock. Despite significant structural similarities between ... More
The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.
The class B type I scavenger receptor, (SR-BI), is a member of the CD36 superfamily of proteins and is a physiologically relevant, high affinity cell surface high density lipoprotein (HDL) receptor that mediates selective lipid uptake. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated ... More
Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand.
AuthorsPampori N, Hato T, Stupack DG, Aidoudi S, Cheresh DA, Nemerow GR, Shattil SJ
JournalJ Biol Chem
PubMed ID10419468
Integrin alpha(V)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(V)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been ... More
Interspecies communication in Streptococcus gordonii-Veillonella atypica biofilms: signaling in flow conditions requires juxtaposition.
AuthorsEgland PG, Palmer RJ, Kolenbrander PE
JournalProc Natl Acad Sci U S A
PubMed ID15546975
During the development of human oral biofilm communities, the spatial arrangement of the bacteria is thought to be driven by metabolic interactions between them. Streptococcus gordonii and Veillonella atypica, two early colonizing members of the dental plaque biofilm, have been postulated to participate in metabolic communication; S. gordonii ferments carbohydrates ... More
Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection.
AuthorsGu X, Lawrence R, Krieger M
JournalJ Biol Chem
PubMed ID10734045
The murine class B, type I scavenger receptor (mSR-BI) is a receptor for both high density lipoprotein (HDL) and low density lipoprotein (LDL) and mediates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to generate mSR-BI mutants in which some, but ... More
Charge- and size-based separation of macromolecules using ultrathin silicon membranes.
Commercial ultrafiltration and dialysis membranes have broad pore size distributions and are over 1,000 times thicker than the molecules they are designed to separate, leading to poor size cut-off properties, filtrate loss within the membranes, and low transport rates. Nanofabricated membranes have great potential in molecular separation applications by offering ... More
Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm.
Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to ... More
Binding and internalization of fluorescent opioid peptide conjugates in living cells.
AuthorsArttamangkul S, Alvarez-Maubecin V, Thomas G, Williams JT, Grandy DK
JournalMol Pharmacol
PubMed ID11093798
The dynamics of agonist-stimulated opioid receptor internalization and trafficking have been difficult to study in living cells in part because the available probes were inadequate. To overcome this obstacle, six new fluorescent opioid peptides were developed. Dermorphin (DERM), deltorphin (DELT), TIPP, and endomorphin were conjugated to BODIPY TR or Alexa ... More
Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution.
AuthorsVan Damme EJ, Nakamura-Tsuruta S, Smith DF, Ongenaert M, Winter HC, Rougé P, Goldstein IJ, Mo H, Kominami J, Culerrier R, Barre A, Hirabayashi J, Peumans WJ
JournalBiochem J
PubMed ID17288538
A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a ... More
Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates.
AuthorsSutherland MS, Sanderson RJ, Gordon KA, Andreyka J, Cerveny CG, Yu C, Lewis TS, Meyer DL, Zabinski RF, Doronina SO, Senter PD, Law CL, Wahl AF
JournalJ Biol Chem
PubMed ID16484228
The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and ... More
Wnt2b controls retinal cell differentiation at the ciliary marginal zone.
AuthorsKubo F, Takeichi M, Nakagawa S
JournalDevelopment
PubMed ID12490564
The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ... More
New insights into extracellular matrix assembly and reorganization from dynamic imaging of extracellular matrix proteins in living osteoblasts.
AuthorsSivakumar P, Czirok A, Rongish BJ, Divakara VP, Wang YP, Dallas SL
JournalJ Cell Sci
PubMed ID16537652
The extracellular matrix (ECM) has been traditionally viewed as a static scaffold that supports cells and tissues. However, recent dynamic imaging studies suggest that ECM components are highly elastic and undergo continual movement and deformation. Latent transforming growth factor beta (TGFbeta) binding protein-1 (LTBP1) is an ECM glycoprotein that binds ... More
PDGFRß-P2A-CreER
AuthorsCuervo H, Pereira B, Nadeem T, Lin M, Lee F, Kitajewski J, Lin CS
JournalAngiogenesis
PubMed ID28752390
Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in ... More
Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.
AuthorsConic S, Desplancq D, Tora L, Weiss E
JournalBio Protoc
PubMed ID30467550
The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous protein in living cells ... More
Targeting the human MUC1-C oncoprotein with an antibody-drug conjugate.
AuthorsPanchamoorthy G, Jin C, Raina D, Bharti A, Yamamoto M, Adeebge D, Zhao Q, Bronson R, Jiang S, Li L, Suzuki Y, Tagde A, Ghoroghchian PP, Wong KK, Kharbanda S, Kufe D
JournalJCI Insight
PubMed ID29925694
Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed on the surface of diverse human carcinomas and is an attractive target for the development of mAb-based therapeutics. However, attempts at targeting the shed MUC1 N-terminal subunit have been unsuccessful. We report here the generation of mAb 3D1 against ... More
Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.
AuthorsConic S, Desplancq D, Ferrand A, Fischer V, Heyer V, Reina San Martin B, Pontabry J, Oulad-Abdelghani M, Babu N K, Wright GD, Molina N, Weiss E, Tora L
JournalJ Cell Biol
PubMed ID29440513
Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing ... More
Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics.
AuthorsDurham ND, Agrawal A, Waltari E, Croote D, Zanini F, Fouch M, Davidson E, Smith O, Carabajal E, Pak JE, Doranz BJ, Robinson M, Sanz AM, Albornoz LL, Rosso F, Einav S, Quake SR, McCutcheon KM, Goo L
JournalElife
PubMed ID31820734
Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. ... More
Exogenous Keratinocyte Growth Factor Is Not Required for Pigmentation of Skin Substitutes with Three Isogeneic Cell Types.
AuthorsBoyce ST, Supp DM, Lloyd CM
JournalTissue Eng Part A
PubMed ID31559928
Engineered skin substitutes (ESS) containing human fibroblasts (hF) and human keratinocytes (hK) provide significant medical benefits for treatment of acute and chronic skin wounds, including, but not limited to, burns, burn scars, congenital skin lesions, and cutaneous ulcers. However, anatomic deficiencies, such as lack of pigment, can contribute to long-term ... More