SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum.
AuthorsZhuo L,Kanamori A,Kannagi R,Itano N,Wu J,Hamaguchi M,Ishiguro N,Kimata K
JournalThe Journal of biological chemistry
PubMed ID16702221
A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes.
AuthorsBeeton C, Wulff H, Singh S, Botsko S, Crossley G, Gutman GA, Cahalan MD, Pennington M, Chandy KG
JournalJ Biol Chem
PubMed ID12511563
T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells ... More
Inhibition of platelet-derived growth factor-BB-induced receptor activation and fibroblast migration by hyaluronan activation of CD44.
AuthorsLi L, Heldin CH, Heldin P
JournalJ Biol Chem
PubMed ID16809345
'The extracellular matrix molecule hyaluronan was found to suppress platelet-derived growth factor (PDGF) beta-receptor activation and PDGF-BB-induced migration of primary human dermal fibroblasts. The suppressive effect of hyaluronan was neutralized by a monoclonal antibody that specifically inhibits hyaluronan binding to its receptor CD44. Moreover, co-immunoprecipitation experiments showed that the PDGF ... More
PKC putative phosphorylation site Ser235 is required for MIP/AQP0 translocation to the plasma membrane.
AuthorsGolestaneh N, Fan J, Zelenka P, Chepelinsky AB,
JournalMol Vis
PubMed ID18523655
'PURPOSE: To investigate the functional significance of MIP/AQP0 phosphorylation at serine(235). METHODS: MIP/AQP0 expression and cellular localization was studied in rat lens epithelia explants induced to differentiate by FGF-2. MIP wild type (WT) and MIP (S235A) mutant expression plasmids were constructed and transiently expressed in RK13 cells. Subcellular localization of ... More
An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression.
AuthorsTrinidad EM, Ballesteros M, Zuloaga J, Zapata A, Alonso-Colmenar LM,
JournalBlood
PubMed ID19828693
'Chronic lymphocytic leukemia (CLL) cell migration into lymphoid tissues is an important aspect of the pathobiology of this disease. Here, we investigated the role of ephrin-A4 (EFNA4) in the transendothelial migration (TEM) capacity of CLL and normal B cells through interacting with endothelial EphA2 (erythropoietin-producing hepatocellular carcinoma). CLL cells showed ... More
Complement C1q is dramatically up-regulated in brain microglia in response to transient global cerebral ischemia.
AuthorsSchäfer MK, Schwaeble WJ, Post C, Salvati P, Calabresi M, Sim RB, Petry F, Loos M, Weihe E
JournalJ Immunol
PubMed ID10799911
'Recent evidence suggests that the pathophysiology of neurodegenerative and inflammatory neurological diseases has a neuroimmunological component involving complement, an innate humoral immune defense system. The present study demonstrates the effects of experimentally induced global ischemia on the biosynthesis of C1q, the recognition subcomponent of the classical complement activation pathway, in ... More
Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.
AuthorsHirsch JD, Eslamizar L, Filanoski BJ, Malekzadeh N, Haugland RP, Beechem JM, Haugland RP
JournalAnal Biochem
PubMed ID12419349
'The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by ... More
Identification of nicotinic acetylcholine receptor recycling and its role in maintaining receptor density at the neuromuscular junction in vivo.
AuthorsBruneau E, Sutter D, Hume RI, Akaaboune M
JournalJ Neurosci
PubMed ID16251443
'In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin ... More
Targeting quantum dots to surface proteins in living cells with biotin ligase.
AuthorsHowarth M, Takao K, Hayashi Y, Ting AY
JournalProc Natl Acad Sci U S A
PubMed ID15897449
'Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a ... More
Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.
AuthorsMullock BM, Smith CW, Ihrke G, Bright NA, Lindsay M, Parkinson EJ, Brooks DA, Parton RG, James DE, Luzio JP, Piper RC
JournalMol Biol Cell
PubMed ID10982406
'Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, ... More
One-step biotinylation procedure for carbohydrates to study carbohydrate-protein interactions.
AuthorsGrün CH, van Vliet SJ, Schiphorst WE, Bank CM, Meyer S, van Die I, van Kooyk Y
JournalAnal Biochem
PubMed ID16713984
'Protein-carbohydrate interactions play crucial roles in numerous biological processes. To study these interactions, we developed a simple and fast procedure for the biotinylation of carbohydrates based on reductive amination. The method allows complete and stable biotinylation of small quantities of oligosaccharides and includes a rapid and simple procedure to remove ... More
Pepsin pretreatment allows collagen IV immunostaining of blood vessels in adult mouse brain.
AuthorsFranciosi S, De Gasperi R, Dickstein DL, English DF, Rocher AB, Janssen WG, Christoffel D, Sosa MA, Hof PR, Buxbaum JD, Elder GA
JournalJ Neurosci Methods
PubMed ID17403541
'While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods ... More
Targeting green fluorescent protein to dendritic membrane in central neurons.
'Dendritic and axonal processes are input and output sites, respectively, of neuronal information, and detailed visualization of these processes may be indispensable for elucidating the neuronal circuits and revealing the principles of neuronal functions. To establish a method for completely visualizing dendritic processes, we first developed green fluorescent protein (GFP)-based ... More
Restriction of receptor movement alters cellular response: physical force sensing by EphA2.
AuthorsSalaita K, Nair PM, Petit RS, Neve RM, Das D, Gray JW, Groves JT,
JournalScience
PubMed ID20223987
'Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, ... More
Versatile, high-resolution anterograde labeling of vagal efferent projections with dextran amines.
'None of the anterograde tracers used to label and investigate vagal preganglionic neurons projecting to the viscera has proved optimal for routine and extensive labeling of autonomic terminal fields. To identify an alternative tracer protocol, the present experiment evaluated whether dextran conjugates, which have produced superior results in the CNS, ... More
ECM regulates MT1-MMP localization with beta1 or alphavbeta3 integrins at distinct cell compartments modulating its internalization and activity on human endothelial cells.
AuthorsGálvez BG, Matías-Román S, Yáñez-Mó M, Sánchez-Madrid F, Arroyo AG
JournalJ Cell Biol
PubMed ID12427871
'Regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) by different extracellular matrices (ECMs) on human endothelial cells (ECs) has been investigated. First, MT1-MMP is found at the intercellular contacts of confluent ECs grown on beta1 integrin-dependent matrix such as type 1 collagen (COL I), fibronectin (FN), or fibrinogen (FG), but not ... More
Collagen XVI harbors an integrin alpha1 beta1 recognition site in its C-terminal domains.
AuthorsEble JA, Kassner A, Niland S, Mörgelin M, Grifka J, Grässel S
JournalJ Biol Chem
PubMed ID16754661
'Collagen XVI is integrated tissue-dependently into distinct fibrillar aggregates, such as D-banded cartilage fibrils and fibrillin-1-containing microfibrils. In skin, the distribution of collagen XVI overlaps that of the collagen-binding integrins alpha1 beta1 and alpha2 beta1. Basal layer keratinocytes express integrin alpha2 beta1, whereas integrin alpha1 beta1 occurs in smooth muscle ... More
Intracellular astrocyte calcium waves in situ increase the frequency of spontaneous AMPA receptor currents in CA1 pyramidal neurons.
AuthorsFiacco TA, McCarthy KD
JournalJ Neurosci
PubMed ID14736858
'Spontaneous neurotransmitter release and activation of group I metabotropic glutamate receptors (mGluRs) each play a role in the plasticity of neuronal synapses. Astrocytes may contribute to short- and long-term synaptic changes by signaling to neurons via these processes. Spontaneous whole-cell AMPA receptor (AMPAR) currents were recorded in CA1 pyramidal cells ... More
Cyclic AMP- and cyclic GMP-dependent protein kinases differ in their regulation of cyclic AMP response element-dependent gene transcription.
AuthorsCollins SP, Uhler MD
JournalJ Biol Chem
PubMed ID10085070
'The ability of cGMP-dependent protein kinases (cGKs) to activate cAMP response element (CRE)-dependent gene transcription was compared with that of cAMP-dependent protein kinases (cAKs). Although both the type Ibeta cGMP-dependent protein kinase (cGKIbeta) and the type II cAMP-dependent protein kinase (cAKII) phosphorylated the cytoplasmic substrate VASP (vasodilator- and A kinase-stimulated ... More
Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.
AuthorsElson-Schwab L, Garner OB, Schuksz M, Crawford BE, Esko JD, Tor Y
JournalJ Biol Chem
PubMed ID17311923
'Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we ... More
Gap junctional communication in the early Xenopus embryo.
AuthorsLandesman Y, Goodenough DA, Paul DL
JournalJ Cell Biol
PubMed ID10953017
'In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of ... More
CD1d-restricted NKT cells: an interstrain comparison.
'CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, ... More
Detection of low-abundance membrane markers by immunofluorescence--a comparison of alternative high-sensitivity methods and reagents.
'The analysis of membrane molecules using antibodies detected by immunofluorescence staining and flow cytometry is used widely in research and diagnostic immunology. Conventional staining techniques readily detect molecules present at concentrations of around 2000 molecules per cell, but some molecules are expressed and function at much lower abundance. We described ... More
Optical measurement of transverse molecular diffusion in a microchannel.
AuthorsKamholz AE, Schilling EA, Yager P
JournalBiophys J
PubMed ID11259309
'Quantitative analysis of molecular diffusion is a necessity for the efficient design of most microfluidic devices as well as an important biophysical method in its own right. This study demonstrates the rapid measurement of diffusion coefficients of large and small molecules in a microfluidic device, the T-sensor, by means of ... More
The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import.
AuthorsWalther TC, Pickersgill HS, Cordes VC, Goldberg MW, Allen TD, Mattaj IW, Fornerod M
JournalJ Cell Biol
PubMed ID12105182
'The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, ... More
Hebbian LTP in feed-forward inhibitory interneurons and the temporal fidelity of input discrimination.
AuthorsLamsa K, Heeroma JH, Kullmann DM
JournalNat Neurosci
PubMed ID15937481
'Cortical information processing requires a delicate balance of excitatory and inhibitory signaling. How is this balance preserved during hippocampal memory encoding, which involves NMDA receptor-dependent long term potentiation (LTP)? This form of LTP occurs at synapses between pyramidal neurons but has not been detected in feed-forward inhibitory interneurons. We show ... More
Deprivation-induced synaptic depression by distinct mechanisms in different layers of mouse visual cortex.
AuthorsCrozier RA, Wang Y, Liu CH, Bear MF
JournalProc Natl Acad Sci U S A
PubMed ID17227847
'Long-term depression (LTD) induced by low-frequency synaptic stimulation (LFS) was originally introduced as a model to probe potential mechanisms of deprivation-induced synaptic depression in visual cortex. In hippocampus, LTD requires activation of postsynaptic NMDA receptors, PKA, and the clathrin-dependent endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It has long been assumed ... More
Expression of the integrin subunit alpha8 in murine lung development.
AuthorsWagner TE, Frevert CW, Herzog EL, Schnapp LM
JournalJ Histochem Cytochem
PubMed ID14500699
'The complex interplay between cells and extracellular matrix (ECM) proteins is critical for lung development. Integrins are key modulators of this interaction. The integrin subunit alpha 8 associates with the beta(1)-subunit to form an RGD-binding integrin. We previously showed that, in adult lung, alpha 8 is expressed in contractile interstitial ... More
Nanoparticles as fluorescence labels: is size all that matters?
AuthorsSwift JL, Cramb DT,
JournalBiophys J
PubMed ID18390610
'Fluorescent labels are often used in bioassays as a means to detect and characterize ligand-receptor binding. This is due in part to the inherently high sensitivity of fluorescence-based technology and the relative accessibility of the technique. There is often little concern raised as to whether or not the fluorescent label ... More
Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv.
AuthorsWang Z, Kim GB, Woo JH, Liu YY, Mathias A, Stavrou S, Neville DM
JournalBioconjug Chem
PubMed ID17352456
'Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the ... More
alpha3alpha5beta2-Nicotinic acetylcholine receptor contributes to the wound repair of the respiratory epithelium by modulating intracellular calcium in migrating cells.
AuthorsTournier JM, Maouche K, Coraux C, Zahm JM, Cloëz-Tayarani I, Nawrocki-Raby B, Bonnomet A, Burlet H, Lebargy F, Polette M, Birembaut P
JournalAm J Pathol
PubMed ID16400009
'Nicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial ... More
Fluorescence-activated cell sorting of specific affibody-displaying staphylococci.
AuthorsWernérus H, Samuelson P, Ståhl S
JournalAppl Environ Microbiol
PubMed ID12957920
'Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using ... More
Targeted cellular delivery of quantum dots loaded on and in biotinylated liposomes.
AuthorsSigot V, Arndt-Jovin DJ, Jovin TM,
JournalBioconjug Chem
PubMed ID20715851
'We describe the preparation, biophysical characterization, and receptor-mediated cellular internalization of biotinylated lipid particles (BLPs) loaded on the surface and internally with two distinct (colors) of quantum dot (QD) probes. BLPs were formulated with 1.4 and 2.7 mol % PEG-lipids containing either a fusogenic or pH-sensitive lipid to promote bilayer ... More
Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis.
'Several mutations that impair the development of blood lineages in the mouse also impair the formation of the lymphatic vasculature and its separation from the blood vasculature. However, the basis for these defects has remained unknown because the mutations characterized affect more than one blood lineage.' ... More
Concentration and separation of proteins in microfluidic channels on the basis of transverse IEF.
AuthorsMacounová K, Cabrera CR, Yager P
JournalAnal Chem
PubMed ID11321320
'The use of microfluidic channels formed by two electrodes made of gold or palladium to perform transverse isoelectric focusing (IEF) is presented as a means for continuous concentration and fractionation of proteins. The microchannels were 40 mm long with an electrode gap of 1.27 mm and a depth of 0.354 ... More
Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.
AuthorsWu P, Shui W, Carlson BL, Hu N, Rabuka D, Lee J, Bertozzi CR,
JournalProc Natl Acad Sci U S A
PubMed ID19202059
'The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the ... 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
Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.
AuthorsEnayetallah AE, French RA, Barber M, Grant DF
JournalJ Histochem Cytochem
PubMed ID16314446
'Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using ... More
Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.
'We report an advanced chemoenzymatic labeling strategy for direct fluorescence detection of O-GlcNAc proteins in gels that facilitates proteomic studies and greatly extend the reach of existing technologies. These new tools also enable the expression and dynamics of O-GlcNAc modifications to be monitored by imaging in cells and tissues. ... More
FRET or no FRET: a quantitative comparison.
AuthorsBerney C, Danuser G
JournalBiophys J
PubMed ID12770904
'Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein ... More
Antibody to Langerin/CD207 localizes large numbers of CD8alpha+ dendritic cells to the marginal zone of mouse spleen.
AuthorsIdoyaga J, Suda N, Suda K, Park CG, Steinman RM,
JournalProc Natl Acad Sci U S A
PubMed ID19168629
Dendritic cells (DCs) are strategically positioned to take up antigens and initiate adaptive immunity. One DC subset expresses CD8alphaalpha in mice and is specialized to capture dying cells and process antigens for MHC class I ... More
Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway.
AuthorsKam NW, Liu Z, Dai H,
JournalAngew Chem Int Ed Engl
PubMed ID16345107
Herein, we present the first systematic investigation of the cellular uptake mechanism and pathway for carbon nanotubes (SWNT). We first show that intracellular transportation of proteins and DNA by SWNTs is indeed general, thus further confirming the transporter ability of these materials. We then present evidence that shows clathrin-dependent endocytosis ... More
Pathogen specific carbohydrate antigen microarrays: a chip for detection of Salmonella O-antigen specific antibodies.
AuthorsBlixt O, Hoffmann J, Svenson S, Norberg T,
JournalGlycoconj J
PubMed ID17558551
A Salmonella O-antigen microarray was developed by covalent coupling of oligosaccharide antigens specific for serogroups Salmonella enterica sv. Paratyphi (group A), Typhimurium (group B) and Enteritidis (group D). Antibodies were correctly detected in sera from patients with culture verified salmonellosis. High serogroup-specificity was seen with the disaccharide antigens. With the ... More
Monitoring of real-time streptavidin-biotin binding kinetics using droplet microfluidics.
AuthorsSrisa-Art M, Dyson EC, deMello AJ, Edel JB,
JournalAnal Chem
PubMed ID18712935
Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin-biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using ... More
Multiplex detection of RNA expression in Drosophila embryos.
AuthorsKosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, Bier E
JournalScience
PubMed ID15297669
We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven differ-ent transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent ... More
Early development of ocular dominance columns.
AuthorsCrowley JC, Katz LC
JournalScience
PubMed ID11082053
The segregation of lateral geniculate nucleus (LGN) axons into ocular dominance columns is believed to involve a prolonged, activity-dependent sorting process. However, visualization of early postnatal ferret LGN axons by direct LGN tracer injections revealed segregated ocular dominance columns <7 days after innervation of layer 4. These early columns were ... More
Transformation of olfactory representations in the Drosophila antennal lobe.
AuthorsWilson RI, Turner GC, Laurent G
JournalScience
PubMed ID14684826
Molecular genetics has revealed a precise stereotypy in the projection of primary olfactory sensory neurons onto secondary neurons. A major challenge is to understand how this mapping translates into odor responses in these second-order neurons. We investigated this question in Drosophila using whole-cell recordings in vivo. We observe that monomolecular ... More
A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.
AuthorsRaja SM, Metkar SS, Höning S, Wang B, Russin WA, Pipalia NH, Menaa C, Belting M, Cao X, Dressel R, Froelich CJ
JournalJ Biol Chem
PubMed ID15788411
The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and ... More
Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species.
AuthorsTóth ZE, Mezey E,
JournalJ Histochem Cytochem
PubMed ID17242468
After immunohistochemistry (IHC) began to be used routinely, a number of investigators worked on methods for staining multiple molecules in the same tissue sections or cells. Achieving this goal was not easy, however. One reason for this is that the majority of primary antibodies used in IHC reactions are raised ... More
Alteration in neonatal nutrition causes perturbations in hypothalamic neural circuits controlling reproductive function.
AuthorsCaron E, Ciofi P, Prevot V, Bouret SG,
JournalJ Neurosci
PubMed ID22895731
It is increasingly accepted that alterations of the early life environment may have lasting impacts on physiological functions. In particular, epidemiological and animal studies have indicated that changes in growth and nutrition during childhood and adolescence can impair reproductive function. However, the precise biological mechanisms that underlie these programming effects ... More
Cannabinoids protect astrocytes from ceramide-induced apoptosis through the phosphatidylinositol 3-kinase/protein kinase B pathway.
AuthorsGómez Del Pulgar T, De Ceballos ML, Guzmán M, Velasco G,
JournalJ Biol Chem
PubMed ID12133838
Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central nervous system by binding to the CB(1) cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However, those studies have been performed in neurons, whereas no ... More
Two-color fluorescence labeling in acrolein-fixed brain tissue.
AuthorsLuquin E, Pérez-Lorenzo E, Aymerich MS, Mengual E,
JournalJ Histochem Cytochem
PubMed ID20051381
Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of ... More
Neuronal classification and marker gene identification via single-cell expression profiling of brainstem vestibular neurons subserving cerebellar learning.
AuthorsKodama T, Guerrero S, Shin M, Moghadam S, Faulstich M, du Lac S,
JournalJ Neurosci
PubMed ID22674258
Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ... 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
Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens.
AuthorsStowell SR, Arthur CM, Mehta P, Slanina KA, Blixt O, Leffler H, Smith DF, Cummings RD,
JournalJ Biol Chem
PubMed ID18216021
Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward ... More
DNA methylation promotes Aurora-B-driven phosphorylation of histone H3 in chromosomal subdomains.
AuthorsMonier K, Mouradian S, Sullivan KF,
JournalJ Cell Sci
PubMed ID17164288
Confinement of enzymatic reactions to nuclear and chromosomal subdomains regulates functional organization of the nucleus. Aurora-B kinase regulates cell-cycle-dependent phosphorylation of chromosomal substrates through sequential localization to a series of sites on chromosomes and the mitotic spindle. In G2 nuclei, Aurora-B recruitment to heterochromatin restricts histone H3S10 phosphorylation to a ... More
Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.
AuthorsHannoush RN, Arenas-Ramirez N,
JournalACS Chem Biol
PubMed ID19505150
Fatty acylation or lipid modification of proteins controls their cellular activation and diverse roles in physiology. It mediates protein-protein and protein-membrane interactions and plays an important role in regulating cellular signaling pathways. Currently, there is need for visualizing lipid modifications of proteins in cells. Herein we report novel chemical probes ... More
Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.
AuthorsPitulescu ME, Schmidt I, Benedito R, Adams RH,
JournalNat Protoc
PubMed ID20725067
The retina is a powerful experimental system for the analysis of angiogenic blood vessel growth in the postnatal organisms. The three-dimensional architecture of the vessel network and processes as diverse as endothelial cell (EC) proliferation, sprouting, perivascular cell recruitment, vessel remodeling or maturation can be investigated at high resolution. The ... More
Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain.
AuthorsVasudevan A, Long JE, Crandall JE, Rubenstein JL, Bhide PG,
JournalNat Neurosci
PubMed ID18344991
Prevailing notions of cerebral vascularization imply that blood vessels sprout passively into the brain parenchyma from pial vascular plexuses to meet metabolic needs of growing neuronal populations. Endothelial cells, building blocks of blood vessels, are thought to be homogeneous in the brain with respect to their origins, gene expression patterns ... More
Giant neurons in the macaque pulvinar: a distinct relay subpopulation.
AuthorsImura K, Rockland KS,
JournalFront Neuroanat
PubMed ID18958196
Calbindin positive (CB+) giant neurons are known to occur within the pulvinar nucleus in subhuman primates. Here, we demonstrate by combined retrograde tracing and immunocytochemistry that at least some of these are pulvinocortical relay neurons, and further report several distinctive features. First, in contrast with non-giant relay neurons, the giant ... More
Heterocellular contact at the myoendothelial junction influences gap junction organization.
AuthorsIsakson BE, Duling BR
JournalCirc Res
PubMed ID15961721
Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated ... More
Identification of a signal peptide for unconventional secretion.
AuthorsDupont E, Prochiantz A, Joliot A
JournalJ Biol Chem
PubMed ID17242404
Homeoproteins are a class of transcription factors defined by the structure of their DNA-binding domain, the homeodomain. In addition to their nuclear cell-autonomous activities, homeoproteins transfer between cells, thanks to two separate steps of secretion and internalization, which both rely on unconventional mechanisms. Internalization is driven by the third helix ... More
Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.
AuthorsBloch W, Fan Y, Han J, Xue S, Schöneberg T, Ji G, Lu ZJ, Walther M, Fässler R, Hescheler J, Addicks K, Fleischmann BK
JournalJ Cell Biol
PubMed ID11514589
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found ... More
Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers.
AuthorsStock RP, Olvera A, Sánchez R, Saralegui A, Scarfì S, Sanchez-Lopez R, Ramos MA, Boffa LC, Benatti U, Alagón A
JournalNat Biotechnol
PubMed ID11231555
Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is ... More
Protection of islets by in situ peptide-mediated transduction of the Ikappa B kinase inhibitor Nemo-binding domain peptide.
AuthorsRehman KK, Bertera S, Bottino R, Balamurugan AN, Mai JC, Mi Z, Trucco M, Robbins PD
JournalJ Biol Chem
PubMed ID12524423
We have previously demonstrated that adenoviral gene transfer of the NF-kappaB inhibitor IkappaB to human islets results in protection from interleukin (IL)-1beta-mediated dysfunction and apoptosis. Here we report that human and mouse islets can be efficiently transduced by a cationic peptide transduction domain (PTD-5) without impairment of islet function. PTD ... More
Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin.
The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated alpha-syntrophin null mice by targeted gene disruption to test the function of this association. The alpha-Syn(-/)- ... More
Localization of endogenous biotin-containing proteins in mouse Bergmann glial cells.
AuthorsYagi T, Terada N, Baba T, Ohno S
JournalHistochem J
PubMed ID14626347
A peroxidase-conjugated avidin-biotin complex was used to detect endogenous biotin-containing proteins in mouse cerebellum. By this method, Bergmann glial cells were found to be strongly labelled in the adult mouse cerebellum. Developmentally, cells in the granular layer, probably astrocytes, appeared to be labelled around postnatal 10-day (P10). Their labelling decreased ... More
Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells.
AuthorsGassama-Diagne A, Yu W, ter Beest M, Martin-Belmonte F, Kierbel A, Engel J, Mostov K
JournalNat Cell Biol
PubMed ID16921364
Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP ... More
Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections.
The poor correlation between cellular immunity to respiratory virus infections and the numbers of memory CD8(+) T cells in the secondary lymphoid organs suggests that there may be additional reservoirs of T cell memory to this class of infection. Here we identify a substantial population of Ag-specific T cells in ... More
Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion.
AuthorsLeikina E, Mittal A, Cho MS, Melikov K, Kozlov MM, Chernomordik LV
JournalJ Biol Chem
PubMed ID15078874
Current models for membrane fusion in diverse biological processes are focused on the local action of fusion proteins present in the contact zone where the proteins anchored in one membrane might interact directly with the other membrane. Are the fusion proteins outside of the contact zone just bystanders? Here we ... More
The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50[middle dot]hMRE11[middle dot]NBS1 complex DNA repair activity.
AuthorsTauchi H, Kobayashi J, Morishima K, Matsuura S, Nakamura A, Shiraishi T, Ito E, Masnada D, Delia D, Komatsu K
JournalJ Biol Chem
PubMed ID11062235
NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation ... More
Peptide tags for labeling membrane proteins in live cells with multiple fluorophores.
AuthorsMcCann CM, Bareyre FM, Lichtman JW, Sanes JR
JournalBiotechniques
PubMed ID16018556
We describe a method to label specific membrane proteins with fluorophores for live imaging. Fusion proteins are generated that incorporate into their extracellular domains short peptide sequences (13-38 amino acids) recognized with high affinity and specificity by protein ligands, alpha-bungarotoxin (BTX), or streptavidin (SA). Many fluorophore- and enzyme-conjugated derivatives of ... More
Calbindin immunoreactivity in the enteric nervous system of larval and adult zebrafish (Danio rerio).
AuthorsOlsson C
JournalCell Tissue Res
PubMed ID21327820
Calbindin is a calcium-binding protein, commonly found in certain subpopulations of the enteric nervous system in mammals. Recently, calbindin-immunoreactive enteric neurons have also been demonstrated in shorthorn sculpin (Myoxocephalus scorpius). In the present study, calbindin immunoreactivity has been investigated in the gut of adult and larval zebrafish (Danio rerio) and ... More
Caspase-cleavage of tau is an early event in Alzheimer disease tangle pathology.
Neurofibrillary tangles (NFTs) are composed of abnormal aggregates of the cytoskeletal protein tau. Together with amyloid beta (Abeta) plaques and neuronal and synaptic loss, NFTs constitute the primary pathological hallmarks of Alzheimer disease (AD). Recent evidence also suggests that caspases are activated early in the progression of AD and may ... More
Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active.
AuthorsBialkowska K, Kulkarni S, Du X, Goll DE, Saido TC, Fox JE
JournalJ Cell Biol
PubMed ID11062268
Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is ... More
Intracellular mislocalization of mutant podocin and correction by chemical chaperones.
AuthorsOhashi T, Uchida K, Uchida S, Sasaki S, Nihei H
JournalHistochem Cell Biol
PubMed ID12649741
The NPHS2 gene encoding the podocin protein was causally linked to the autosomal recessive type of steroid-resistant nephrotic syndrome. In this study, we investigated the consequence of the R138Q mutation of podocin, one of the most common missense mutations in the NPHS2 gene, by examining the expression of the wild-type ... More
Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence.
AuthorsSchmid I, Cole SW, Korin YD, Zack JA, Giorgi JV
JournalCytometry
PubMed ID10679728
BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: ... More
Detection of two different mRNAs in a single section by dual in situ hybridization: a comparison between colorimetric and fluorescent detection.
AuthorsBarroso-Chinea P, Aymerich MS, Castle MM, Pérez-Manso M, Tuñón T, Erro E, Lanciego JL
JournalJ Neurosci Methods
PubMed ID17306886
We have compared the performance of two methods designed to simultaneously detect two different mRNAs within a single brain section by dual ISH. Specific mRNA riboprobes labeled with biotin and digoxigenin were simultaneously hybridized and visualized using either brightfield or fluorescence microscopy. For brightfield visualization, the biotin-labeled riboprobe was detected ... More
Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent ... More
Identification and functions of chondroitin sulfate in the milieu of neural stem cells.
AuthorsIda M, Shuo T, Hirano K, Tokita Y, Nakanishi K, Matsui F, Aono S, Fujita H, Fujiwara Y, Kaji T, Oohira A
JournalJ Biol Chem
PubMed ID16373347
The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular ... More
Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells.
AuthorsPoliak S, Matlis S, Ullmer C, Scherer SS, Peles E
JournalJ Cell Biol
PubMed ID12403818
The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of ... More
Collagen-bound von Willebrand factor has reduced affinity for factor VIII.
AuthorsBendetowicz AV, Wise RJ, Gilbert GE
JournalJ Biol Chem
PubMed ID10212199
von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether ... More
The tyrp1-Tag/tyrp1-FGFR1-DN bigenic mouse: a model for selective inhibition of tumor development, angiogenesis, and invasion into the neural tissue by blockade of fibroblast growth factor receptor activity.
We describe herein a new transgenic mouse tumor model in which fibroblast growth factor (FGF) receptor activity is selectively inhibited. Tyrp1-Tag mice that develop early vascularized tumors of the retinal pigment epithelium were crossed with tyrp1-FGFR1-DN mice that express dominant-negative FGF receptors in the retinal pigment epithelium to generate bigenic ... More
Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants.
AuthorsKellar KL, Kalwar RR, Dubois KA, Crouse D, Chafin WD, Kane BE
JournalCytometry
PubMed ID11598944
BACKGROUND: An increasing volume of data suggests a relationship between cytokine levels in human body fluids and disease pathogenesis. Traditionally, many individual assays would be performed to measure the large number of known cytokines and determine their associations with disease. A new technique for the simultaneous measurement of multiple cytokines ... More
Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor.
AuthorsMorel Y, Schiano de Colella JM, Harrop J, Deen KC, Holmes SD, Wattam TA, Khandekar SS, Truneh A, Sweet RW, Gastaut JA, Olive D, Costello RT
JournalJ Immunol
PubMed ID11035077
The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this ... More
Infection of human airway epithelium by human and avian strains of influenza a virus.
We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that ... More
Anterograde tracing and immunohistochemical characterization of potentially mechanosensitive vagal afferents in the esophagus.
AuthorsKressel M, Radespiel-Tröger M
JournalJ Comp Neurol
PubMed ID10440717
Vagal mechanosensitive afferents with an important functional role in esophageal peristalsis are well known from physiological studies. It is not known whether these fibers represent a separate subpopulation among all vagal afferents projecting to the esophageal wall. A morphological and immunohistochemical description of vagal afferents was undertaken to define their ... More
Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content.
AuthorsSchmid I, Dagarag MD, Hausner MA, Matud JL, Just T, Effros RB, Jamieson BD
JournalCytometry
PubMed ID12442309
BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate ... More
AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia.
AuthorsNoda M, Nakanishi H, Nabekura J, Akaike N
JournalJ Neurosci
PubMed ID10627602
Microglial cells were isolated from rat cerebral cortex, and kainate (KA)-induced inward current was measured at a holding potential of -40 or -60 mV. 6-Cyano-7-nitroquinoxaline-2, 3-dione-sensitive KA-induced currents increased with increasing KA concentration. The half-activation concentration and Hill coefficient were 3.3 x 10(-4) M and 1.4, respectively. Although glutamate (Glu) ... More
Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry.
AuthorsSchmid I, Cole SW, Zack JA, Giorgi JV
JournalJ Immunol Methods
PubMed ID10675764
We developed a method for simultaneous flow cytometric analysis of three-color immunofluorescence and DNA content. We show here that staining with 7-amino-actinomycin D (7-AAD) at 10 microg/ml using a phosphate-citrate buffer at low pH containing saponin for cell membrane permeabilization yields good resolution DNA histograms with low coefficients of variation. ... More
A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells.
AuthorsAlbarran B, To R, Stayton PS
JournalProtein Eng Des Sel
PubMed ID15820981
The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a ... More
Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper.
AuthorsSyme CA, Hamilton KL, Jones HM, Gerlach AC, Giltinan L, Papworth GD, Watkins SC, Bradbury NA, Devor DC
JournalJ Biol Chem
PubMed ID12493744
We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted ... More