Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms.
AuthorsMarg A, Meyer T, Vigneron M, Vinkemeier U,
JournalCytometry A
PubMed ID18773464
'The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the ... 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
The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane.
AuthorsJacobson C, Côté PD, Rossi SG, Rotundo RL, Carbonetto S
JournalJ Cell Biol
PubMed ID11157973
'The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve-muscle synapses. We ... More
Larval Schistosoma mansoni excretory-secretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria glabrata (Gastropoda) via surface carbohydrate binding receptors.
AuthorsJohnston LA, Yoshino TP
JournalJ Parasitol
PubMed ID11534642
'Flow cytometric analysis of circulating blood cells (hemocytes) of Biomphalaria glabrata, molluscan intermediate host of Schistosoma mansoni, revealed the presence of 2 overlapping hemocyte subpopulations, designated R1 and R2. R1 hemocytes are characterized by their smaller size, reduced granularity, and the presence of the BGH1 surface epitope, whereas R2 cells ... More
Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages.
AuthorsPorte F, Liautard JP, Köhler S
JournalInfect Immun
PubMed ID10417172
'Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence ... More
Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan.
AuthorsBeaven AE, Paynter KT
JournalBiol Bull
PubMed ID10065529
'Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH ... More
A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells.
AuthorsHama Y, Urano Y, Koyama Y, Bernardo M, Choyke PL, Kobayashi H
JournalBioconjug Chem
PubMed ID17105220
'In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away ... More
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
Sickle red blood cells accumulate in tumor.
AuthorsBrown SL, Ewing JR, Nagaraja TN, Swerdlow PS, Cao Y, Fenstermacher JD, Kim JH
JournalMagn Reson Med
PubMed ID14648568
'The preferential accumulation of sickle blood cells in tumor vasculature is demonstrated noninvasively using MRI and sickle red blood cells loaded with Gd-DTPA and invasively by two other techniques. The distribution of red blood cells in rat brain tumors relative to normal brains were measured using three separate techniques: MRI ... More
Thermodynamics of p53 binding to hdm2(1-126): effects of phosphorylation and p53 peptide length.
AuthorsLai Z, Auger KR, Manubay CM, Copeland RA
JournalArch Biochem Biophys
PubMed ID11032416
'Upon exposure to DNA-damaging agents, the p53 tumor suppressor protein is stabilized and activated, leading to cell cycle arrest, DNA repair, or apoptosis. One of the major factors that regulates the level and the transcriptional activity of p53 is the hdm2 oncoprotein. hdm2 binds to the N-terminal transactivation domain of ... More
Yeast protein farnesyltransferase. pKas of peptide substrates bound as zinc thiolates.
AuthorsRozema DB, Poulter CD
JournalBiochemistry
PubMed ID10529185
'Protein farnesyltransferase (PFTase) is a zinc metalloenzyme that catalyzes the posttranslational alkylation of the cysteine in C-terminal -Ca(1)a(2)X sequences by a 15-carbon farnesyl residue, where C is cysteine, a(1) and a(2) are normally aliphatic amino acids, and X is an amino acid that specifies selectivity for the farnesyl moiety. Formation ... More
Changes in root cap pH are required for the gravity response of the Arabidopsis root.
AuthorsFasano JM, Swanson SJ, Blancaflor EB, Dowd PE, Kao TH, Gilroy S
JournalPlant Cell
PubMed ID11283344
'Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a ... More
Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport.
'Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in ... More
Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis.
AuthorsBernardo J, Long HJ, Simons ER,
JournalCytometry A
PubMed ID19937952
'Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within ... More
Distribution and quantification of polyethylenimine oligodeoxynucleotide complexes in human skin after iontophoretic delivery using confocal scanning laser microscopy.
AuthorsBrus C, Santi P, Colombo P, Kissel T
JournalJ Control Release
PubMed ID12468220
'Iontophoresis may be a potentially useful technique for the delivery of oligonucleotides into the skin. To enhance intracellular uptake during iontophoresis, we investigated the dermal delivery of oligodeoxynucleotides (ODN) as a polyelectrolyte complex with polyethylenimine (PEI). Perpendicular cross-sectioning was performed to visualize and quantify the penetration properties of double labeled ... More
Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.
AuthorsBarker SL, Clark HA, Swallen SF, Kopelman R, Tsang AW, Swanson JA
JournalAnal Chem
PubMed ID10330907
'Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c'', a hemoprotein known to bind nitric oxide selectively. The cytochrome c'' is labeled with a fluorescent reporter dye, and changes in this dye''s intensity or fluorescence lifetime are observed as the protein ... More
Design and synthesis of a bimodal target-specific contrast agent for angiogenesis.
AuthorsDirksen A, Langereis S, de Waal BF, van Genderen MH, Meijer EW, de Lussanet QG, Hackeng TM
JournalOrg Lett
PubMed ID15606084
'[structure: see text] A bimodal target-specific contrast agent based on a cyclic peptide containing the target-specific NGR sequence, gadolinium(III) diethylenetriaminepentaacetic acid (Gd(III)DTPA), and Oregon Green 488 (OG488) suitable for both MR imaging and optical imaging of angiogenesis is developed. The synthetic strategy for this target-specific contrast agent exploits the use ... More
H-type dimer formation of fluorophores: a mechanism for activatable, in vivo optical molecular imaging.
AuthorsOgawa M, Kosaka N, Choyke PL, Kobayashi H,
JournalACS Chem Biol
PubMed ID19480464
'In vivo molecular imaging with target-specific activatable '
Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor activity.
AuthorsSingh LS, Bukys MA, Beck DO, Kalafatis M
JournalJ Biol Chem
PubMed ID12738785
'We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4') contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor ... 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
Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate.
AuthorsWee KE, Lai Z, Auger KR, Ma J, Horiuchi KY, Dowling RL, Dougherty CS, Corman JI, Wynn R, Copeland RA
JournalJ Protein Chem
PubMed ID11195973
'We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 ... More
Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers.
AuthorsYoo H, Juliano RL
JournalNucleic Acids Res
PubMed ID11058121
'PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer-nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. ... More
Fluorescence quenching: A tool for single-molecule protein-folding study.
AuthorsZhuang X, Ha T, Kim HD, Centner T, Labeit S, Chu S
JournalProc Natl Acad Sci U S A
PubMed ID11121030
'By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from ... More
Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323-331 of factor V represent an interactive site for activated factor X.
AuthorsKalafatis M, Beck DO
JournalBiochemistry
PubMed ID12379114
'We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va ... More
Porous functionalised silica particles: a potential platform for biomolecular screening.
AuthorsJohnston AP, Battersby BJ, Lawrie GA, Trau M,
JournalChem Commun (Camb)
PubMed ID15700057
Novel, porous, functionalised silica particles have been developed with controlled morphology, which promote covalent attachment of fluorescent dyes which can act as an optical barcode.
Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane.
AuthorsTriantafilou M, Morath S, Mackie A, Hartung T, Triantafilou K
JournalJ Cell Sci
PubMed ID15286178
The innate immune system utilises pattern recognition receptors in order to recognise microbial conserved molecular patterns. The family of Toll-like receptors (TLRs) has been shown to act as the main pattern recognition receptors for the innate immune system. Using biochemical as well as fluorescence imaging techniques, TLR2 and TLR4 were ... 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
Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery.
AuthorsGodbey WT, Wu KK, Mikos AG
JournalProc Natl Acad Sci U S A
PubMed ID10220439
Poly(ethylenimine) (PEI) is one of a number of polycations that has been used successfully to transfer genes into living cells. Although PEI shows promise in the field of gene therapy, to date no rigorous proof of mechanism has been published regarding the fate of PEI/DNA administered for transfection. Here we ... More
Bidirectional translocation of neurofilaments along microtubules mediated in part by dynein/dynactin.
Neuronal cytoskeletal elements such as neurofilaments, F-actin, and microtubules are actively translocated by an as yet unidentified mechanism. This report describes a novel interaction between neurofilaments and microtubule motor proteins that mediates the translocation of neurofilaments along microtubules in vitro. Native neurofilaments purified from spinal cord are transported along microtubules ... More
Unimpaired lysosomal acidification in respiratory epithelial cells in cystic fibrosis.
AuthorsHaggie PM, Verkman AS,
JournalJ Biol Chem
PubMed ID19136560
The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., ... More
Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.
Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, ... More
Spectral fluorescence molecular imaging of lung metastases targeting HER2/neu.
AuthorsKoyama Y, Hama Y, Urano Y, Nguyen DM, Choyke PL, Kobayashi H,
JournalClin Cancer Res
PubMed ID17504994
PURPOSE: Surgical resection of pulmonary metastases is now a clinically accepted cancer therapy but its success depends on the accurate localization and removal of all tumor foci. To enhance the detection of pulmonary metastases during surgery, we developed an i.v. administered optical probe that uses a monoclonal antibody, Herceptin (trastuzumab), ... More
Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy.
AuthorsMegens RT, Oude Egbrink MG, Cleutjens JP, Kuijpers MJ, Schiffers PH, Merkx M, Slaaf DW, van Zandvoort MA,
JournalMol Imaging
PubMed ID17711780
We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), ... More
Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis.
AuthorsBailey RW, Olson ED, Vu MP, Brueseke TJ, Robertson L, Christensen RE, Parker KH, Judd AM, Bell JD,
JournalBiophys J
PubMed ID17545239
During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis ... More
An affinity-based method for the purification of fluorescently-labeled biomolecules.
AuthorsNguyen T, Joshi NS, Francis MB
JournalBioconjug Chem
PubMed ID16848391
Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the ... More
Fibroblast growth factor receptor-1 interacts with the T-cell receptor signalling pathway.
AuthorsByrd VM, Kilkenny DM, Dikov MM, Reich MB, Rocheleau JV, Armistead WJ, Thomas JW, Miller GG
JournalImmunol Cell Biol
PubMed ID14636241
Fibroblast growth factor receptors are expressed by some T cells, and provide costimulation for these cells. Such receptors allow T cells to respond to fibroblast growth factors expressed in response to injury and inflammation and may provide a mechanism for 'context-dependent' responses to antigens within the local microenvironment. The mechanisms ... More
Development and cellular applications of fiber optic nitric oxide sensors based on a gold-adsorbed fluorophore.
AuthorsBarker SL, Kopelman R
JournalAnal Chem
PubMed ID9852778
A new design for optochemical sensors has been applied to the development of a nitric oxide selective fiber optic sensor. This sensor is composed of a fluorescein derivative dye attached to colloidal gold. The fluorescein dye rearranges as nitric oxide adsorbs onto the gold, inducing a decrease in the fluorescence ... More
Regulation of cell migration by the calcium-dependent protease calpain.
AuthorsHuttenlocher A, Palecek SP, Lu Q, Zhang W, Mellgren RL, Lauffenburger DA, Ginsberg MH, Horwitz AF
JournalJ Biol Chem
PubMed ID9407041
Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain ... More
Fluorescently-labeled fimbrin decorates a dynamic actin filament network in live plant cells.
AuthorsKovar DR, Gibbon BC, McCurdy DW, Staiger CJ
JournalPlanta
PubMed ID11506361
Recently it has been established, through a detailed biochemical analysis, that recombinant Arabidopsis thaliana fimbrin 1 (AtFim1) is a member of the fimbrin/plastin family of actin filament bundling or cross-linking proteins [D.R. Kovar et al. (2000) Plant J 24:625-636]. To determine whether AtFim1 can function as an F-actin-binding protein in ... More
Synthesis and application of submicrometer fluorescence sensing particles for lysosomal pH measurements in murine macrophages.
AuthorsJi J, Rosenzweig N, Griffin C, Rosenzweig Z
JournalAnal Chem
PubMed ID10952534
Phagocytosis of bioparticles such as bacteria and viruses by macrophages is a critical component of the immune response against infections. In this paper we describe the synthesis of submicrometer fluorescent particles with pH sensing capability. The particles are used to measure the pH and to monitor the effect of chloroquine, ... More
Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy.
AuthorsToivola J, Michel PO, Gilbert L, Lahtinen T, Marjomäki V, Hedman K, Vuento M, Oker-Blom C
JournalBiol Chem
PubMed ID14977050
Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of ... More
A real-time fluorescence method to monitor the melting of duplex DNA during transcription initiation by RNA polymerase.
AuthorsMatlock DL, Heyduk T
JournalAnal Biochem
PubMed ID10328775
The melting of duplex DNA in the vicinity of the transcription start site is an essential step of transcription initiation. Here we describe a fluorescent promoter technique which allows the melting of promoter DNA to be observed in a real-time manner with high sensitivity. We have constructed a 114-bp lacUV5 ... More
Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing.
AuthorsNunnally BK, He H, Li LC, Tucker SA, McGown LB
JournalAnal Chem
PubMed ID9212706
Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of ... More
Phagosomal pH determination by dual fluorescence flow cytometry.
AuthorsVergne I, Constant P, Lanéelle G
JournalAnal Biochem
PubMed ID9448851
Several methods have been developed to measure the pH of phagosomes, using fluorescein derivatives as reporter of pH, and spectrofluorimetry, fluorescence microscopy, or flow cytometry as quantification technique. All have major disadvantages, including either a slow or inaccurate response. In the present study, pH determination was achieved on J774-cell phagosomes ... More
2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.
AuthorsStaudt T, Lang MC, Medda R, Engelhardt J, Hell SW
JournalMicrosc Res Tech
PubMed ID17131355
The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 micro m deep into the specimen, the refractive index mismatch results in a ... More
Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes.
AuthorsCowan DB, Noria S, Stamm C, Garcia LM, Poutias DN, del Nido PJ, McGowan FX
JournalCirc Res
PubMed ID11249872
We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be internalized to facilitate endotoxin-dependent signal activation in cardiac myocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell line. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized ... More
Cellular applications of a sensitive and selective fiber-optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase.
AuthorsBarker SL, Zhao Y, Marletta MA, Kopelman R
JournalAnal Chem
PubMed ID10366889
Nitric oxide-selective sensors have been prepared with the heme domain of soluble guanylate cyclase (sGC), the only known receptor for signal transduction involving nitric oxide. Expressed in and purified from E. coli, the heme domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical ... More
Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.
AuthorsKrahn KN, Bouten CV, van Tuijl S, van Zandvoort MA, Merkx M
JournalAnal Biochem
PubMed ID16476406
Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains ... More
Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcgammaRIIA receptors.
AuthorsDowney GP, Botelho RJ, Butler JR, Moltyaner Y, Chien P, Schreiber AD, Grinstein S
JournalJ Biol Chem
PubMed ID10497205
Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such ... More
Capillary electrophoretic analysis of mu- and m-calpain using fluorescently labeled casein substrates.
AuthorsGu X, Whipple-VanPatter G, O'Dwyer M, Zeece M
JournalElectrophoresis
PubMed ID11504070
Calpains are unique calcium-dependent thiol proteases that have been proposed to participate in a number of physiological processes including signal transduction and protein turnover in skeletal muscle. Calpains exist in two major forms. Interestingly, the two forms of protease show no significant difference in their action on various substrates. The ... More
Site-directed mutations of T4 helicase loading protein (gp59) reveal multiple modes of DNA polymerase inhibition and the mechanism of unlocking by gp41 helicase.
AuthorsNelson SW, Yang J, Benkovic SJ
JournalJ Biol Chem
PubMed ID16407253
The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a ... 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
Inhibition of the calcium-dependent tyrosine kinase (CADTK) blocks monocyte spreading and motility.
Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p125(FAK)) but activate a second member of this kinase family, calcium-dependent tyrosine kinase (CADTK; also known as Pyk2/CAKbeta/RAFTK/FAK2), upon adhesion or stimulation with chemokines. To study the role of CADTK in monocyte adherence and motility, we performed immunocytochemical localization that showed ... More
Comparison of the endocytic properties of linear and branched PEIs, and cationic PAMAM dendrimers in B16f10 melanoma cells.
AuthorsSeib FP, Jones AT, Duncan R
JournalJ Control Release
PubMed ID17210200
Many different polymers and architectures are now being developed as polymer therapeutics and non-viral vectors for cytosolic delivery, and cationic dendrimers, and linear and branched poly(ethylenimine)s (PEIs) have been widely used. For rational design and safe transfer into the clinic, it is important to better understand the cellular pharmacokinetics of ... More