Identification, cloning, and characterization of two N-acetylgalactosamine-binding lectins from the albumen gland of Helix pomatia.
AuthorsMarkiv A, Peiris D, Curley GP, Odell M, Dwek MV,
JournalJ Biol Chem
PubMed ID21372134
Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant ... More
Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs.
AuthorsKosturko LD, Maggipinto MJ, Korza G, Lee JW, Carson JH, Barbarese E
JournalMol Biol Cell
PubMed ID16775011
'Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of ... 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
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
Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging.
'Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region ... More
Synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-DNA conjugates with different carbohydrate presentation.
AuthorsUgarte-Uribe B, Pérez-Rentero S, Lucas R, Aviñó A, Reina JJ, Alkorta I, Eritja R, Morales JC,
JournalBioconjug Chem
PubMed ID20590107
'Oligonucleotide conjugates carrying carbohydrates at the 5''-end have been prepared. Glucose, fucose, and saccharides containing glucose at the nonreducing end were attached to DNA strands using the classical phosphoramidite chemistry. Two types of spacers and a dendron scaffold helped to obtain a diversity of sugar presentations in the DNA conjugates. ... More
In vivo passage of albumin from the aqueous humor into the lens.
AuthorsSabah JR, Davidson H, McConkey EN, Takemoto L
JournalMol Vis
PubMed ID15073582
'PURPOSE: To determine if albumin, the major protein component of the aqueous humor, passes into the lens in vivo. METHODS: Rat albumin was covalently-labeled with Alexa 488 fluorophore, purified by gel permeation chromatography, then injected into the aqueous chamber of living rats. At 5 min postinjection, lenses were removed and ... More
Toxicity of organic fluorophores used in molecular imaging: literature review.
AuthorsAlford R, Simpson HM, Duberman J, Hill GC, Ogawa M, Regino C, Kobayashi H, Choyke PL,
JournalMol Imaging
PubMed ID20003892
'Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of ... More
Optical properties of Alexa 488 and Cy5 immobilized on a glass surface.
AuthorsWang L, Gaigalas AK, Reipa V
JournalBiotechniques
PubMed ID15679095
'The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret ... 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
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
Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold.
AuthorsSanchez JF, Lescar J, Chazalet V, Audfray A, Gagnon J, Alvarez R, Breton C, Imberty A, Mitchell EP
JournalJ Biol Chem
PubMed ID16704980
'Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates ... More
HLA-DM constrains epitope selection in the human CD4 T cell response to vaccinia virus by favoring the presentation of peptides with longer HLA-DM-mediated half-lives.
AuthorsYin L, Calvo-Calle JM, Dominguez-Amorocho O, Stern LJ,
JournalJ Immunol
PubMed ID22966084
'HLA-DM (DM) is a nonclassical MHC class II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during Ag presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of ... 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
In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5.
AuthorsOhrt T, Merkle D, Birkenfeld K, Echeverri CJ, Schwille P
JournalNucleic Acids Res
PubMed ID16522647
'Two types of short double-stranded RNA molecules, namely microRNAs (miRNAs) and short interfering RNAs (siRNAs), have emerged recently as important regulators of gene expression. Although these molecules show similar sizes and structural features, the mechanisms of action underlying their respective target silencing activities appear to differ: siRNAs act primarily through ... More
A human CD4+ T cell epitope in the influenza hemagglutinin is cross-reactive to influenza A virus subtypes and to influenza B virus.
AuthorsBabon JA, Cruz J, Ennis FA, Yin L, Terajima M,
JournalJ Virol
PubMed ID22718815
'The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope ... More
Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.
'HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). ... More
X-ray crystal structure of the streptococcal specific phage lysin PlyC.
AuthorsMcGowan S, Buckle AM, Mitchell MS, Hoopes JT, Gallagher DT, Heselpoth RD, Shen Y, Reboul CF, Law RH, Fischetti VA, Whisstock JC, Nelson DC,
JournalProc Natl Acad Sci U S A
PubMed ID22807482
'Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that ... More
Phage-based molecular probes that discriminate force-induced structural states of fibronectin in vivo.
'Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin ' ... More
Fast STED microscopy with continuous wave fiber lasers.
We report on fast beam-scanning stimulated-emission-depletion (STED) microscopy in the visible range using for resolution enhancement compact, low cost and turn-key continuous wave (CW) fiber lasers emitting at 592 nm. Spatial resolutions of 35 to 65 nm in the focal plane are shown for various samples including fluorescent nanoparticles, immuno-stained ... More
Inhibitory Fc?RIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity.
AuthorsVaughan AT, Iriyama C, Beers SA, Chan CH, Lim SH, Williams EL, Shah V, Roghanian A, Frendéus B, Glennie MJ, Cragg MS,
Journal
PubMed ID24227819
A major feature that distinguishes type I from type II anti-CD20 monoclonal antibodies (mAbs) and reduces their therapeutic efficacy is the tendency to internalize from the cell surface. We have shown previously that the extent of internalization correlates with the capacity of type I mAb to simultaneously engage both CD20 ... More
Human CD4+ T cell response to human herpesvirus 6.
AuthorsNastke MD, Becerra A, Yin L, Dominguez-Amorocho O, Gibson L, Stern LJ, Calvo-Calle JM,
JournalJ Virol
PubMed ID22357271
Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline ... More
Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands.
A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high ... More
Surfactant protein-A and toll-like receptor-4 modulate immune functions of preterm baboon lung dendritic cell precursor cells.
AuthorsAwasthi S, Madhusoodhanan R, Wolf R,
JournalCell Immunol
PubMed ID21439559
Lung infections are important risk factors for an increased morbidity and mortality in prematurely-delivered babies. Immaturity of the innate immune components makes them extremely susceptible to infection. Recently, we isolated lung dendritic cell (DC)-precursor cells from preterm fetal baboons. The isolated cells were found to be defective in phagocytosing Escherichia ... More
Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition.
The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and ... More
A FRET-based method to study protein thiol oxidation in histological preparations.
AuthorsMastroberardino PG, Orr AL, Hu X, Na HM, Greenamyre JT,
JournalFree Radic Biol Med
PubMed ID18620047
Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling ... 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
Physiological regulation of yeast cell death in multicellular colonies is triggered by ammonia.
AuthorsVáchová L, Palková Z
JournalJ Cell Biol
PubMed ID15939758
The existence of programmed cell death (PCD) in yeast and its significance to simple unicellular organisms is still questioned. However, such doubts usually do not reflect the fact that microorganisms in nature exist predominantly within structured, multicellular communities capable of differentiation, in which a profit of individual cells is subordinated ... More
Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex.
AuthorsAn S, Musier-Forsyth K
JournalJ Biol Chem
PubMed ID16087664
Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae ... More
Interaction between a 54-kilodalton mammalian cell surface protein and cowpea mosaic virus.
AuthorsKoudelka KJ, Rae CS, Gonzalez MJ, Manchester M
JournalJ Virol
PubMed ID17121801
Cowpea mosaic virus (CPMV), a plant virus that is a member of the picornavirus superfamily, is increasingly being used for nanotechnology applications, including material science, vascular imaging, vaccine development, and targeted drug delivery. For these applications, it is critical to understand the in vivo interactions of CPMV within the mammalian ... More
Functional association between three archaeal aminoacyl-tRNA synthetases.
AuthorsPraetorius-Ibba M, Hausmann CD, Paras M, Rogers TE, Ibba M
JournalJ Biol Chem
PubMed ID17158871
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) ... 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
Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis.
Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog ... 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
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