'The development of allergic asthma is influenced by both genetic and environmental factors. Epidemiologic data often show no clear relationship between the levels of allergen and clinical symptoms. Recent data suggest that bacterial LPS may be a risk factor related to asthma severity. Airborne LPS is typically present at levels ... More
Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate.
AuthorsMartin K, Hart C, Schulenberg B, Jones L, Patton WF
JournalProteomics
PubMed ID11987124
'A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all ... More
Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH.
AuthorsKim JR, Yoon HW, Kwon KS, Lee SR, Rhee SG
JournalAnal Biochem
PubMed ID10906242
'A procedure for detecting proteins that contain H(2)O(2)-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H(2)O(2) in cells. The procedure is based on the facts that H(2)O(2) and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a ... More
The FITC-anti-FITC system is a sensitive alternative to biotin-streptavidin in ELISA.
AuthorsHarmer IJ, Samuel D
JournalJ Immunol Methods
PubMed ID2503563
'In a model ELISA system alkaline phosphatase (AP) absorbed onto microtitre wells was employed as the target antigen. The antigen was then reacted with a monoclonal antibody to AP either unlabelled or labelled with (a) FITC and (b) biotin. The bound anti-AP was then detected with horseradish peroxidase (HRP) conjugates ... More
Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors.
AuthorsSekine-Aizawa Y, Huganir RL,
JournalProc Natl Acad Sci U S A
PubMed ID15563595
'alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate excitatory synaptic transmission and are dynamically regulated during synaptic plasticity in the CNS. The membrane trafficking of AMPA receptors to synapses is critical for the regulation of the efficacy of excitatory synaptic transmission. Direct imaging of AMPA receptors in various cell compartments is important to dissecting ... More
Immunocytochemical localization of the NPY/PYY Y1 receptor in enteric neurons, endothelial cells, and endocrine-like cells of the rat intestinal tract.
AuthorsJackerott M, Larsson LI
JournalJ Histochem Cytochem
PubMed ID9389767
'Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that are considered to mediate inhibitory actions on gastrointestinal motility, secretion, and blood flow. Several receptor subtypes for these peptides have been identified and the Y1, Y2, Y4/PP1, Y5, and Y5/PP2/Y2b receptors have been cloned. In this article we ... More
Colocalization and redistribution of dishevelled and actin during Wnt-induced mesenchymal morphogenesis.
AuthorsTorres MA, Nelson WJ
JournalJ Cell Biol
PubMed ID10871283
'Activation of the Wnt signaling pathway is important for induction of gene expression and cell morphogenesis throughout embryonic development. We examined the subcellular localization of dishevelled, the immediate downstream component from the Wnt receptor, in the embryonic mouse kidney. Using immunofluorescence staining, confocal microscopy, and coimmunoprecipitation experiments, we show that ... More
The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling.
AuthorsMielenz D, Hapke S, Pöschl E, von Der Mark H, von Der Mark K
JournalJ Biol Chem
PubMed ID11278916
'The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the ... More
Syntaxin 7 mediates endocytic trafficking to late endosomes.
AuthorsNakamura N, Yamamoto A, Wada Y, Futai M
JournalJ Biol Chem
PubMed ID10692457
'The lysosome functions are ensured by accurate membrane trafficking in the cell. We found that mouse syntaxin 7 could complement yeast vam3 and pep12 mutants defective in docking/fusion to vacuolar and prevacuolar membranes, respectively. Immunohistochemical studies showed that syntaxin 7 is localized to late endosomes, but not to early endosomes. ... More
IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines.
AuthorsAlbanesi C, Scarponi C, Sebastiani S, Cavani A, Federici M, De Pità O, Puddu P, Girolomoni G
JournalJ Immunol
PubMed ID10903743
'IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, ... More
Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences.
AuthorsZimmer G, Budz L, Herrler G
JournalJ Biol Chem
PubMed ID11418598
'The F (fusion) protein of the respiratory syncytial viruses is synthesized as an inactive precursor F(0) that is proteolytically processed at the multibasic sequence KKRKRR(136) into the subunits F(1) and F(2) by the cellular protease furin. This maturation process is essential for the F protein to gain fusion competence. We ... More
Projection subtypes of rat neostriatal matrix cells revealed by intracellular injection of biocytin.
AuthorsKawaguchi Y, Wilson CJ, Emson PC
JournalJ Neurosci
PubMed ID1698947
'Individual neostriatal-matrix spiny neurons were stained intracellularly with biocytin after intracellular recording in vivo, and their axons were traced into the globus pallidus (GP), entopeduncular nucleus (EP), and/or substantia nigra (SN). The locations of the neurons within the matrix compartment of the neostriatum (NS) were established by immunocytochemical counterstaining of ... More
Functional in vivo MHC class II loading by endogenously synthesized glycoprotein during viral infection.
AuthorsOxenius A, Bachmann MF, Mathis D, Benoist C, Zinkernagel RM, Hengartner H
JournalJ Immunol
PubMed ID9190921
'MHC class II presentation of antigenic peptides derived from soluble proteins is usually preceded by antigenic uptake via (nonreceptor-mediated) endocytosis by professional APCs, followed by processing in endosomal compartments. Although in vitro alternative pathways for MHC class II loading have been described for certain intracellularly synthesized proteins, the importance of ... More
Extracellular DsbA-insensitive folding of Escherichia coli heat-stable enterotoxin STa in vitro.
AuthorsBatisson I, der Vartanian M
JournalJ Biol Chem
PubMed ID10744753
'To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able ... More
Enteric infection acts as an adjuvant for the response to a model food antigen.
AuthorsShi HN, Liu HY, Nagler-Anderson C
JournalJ Immunol
PubMed ID11086051
'Oral administration of soluble protein Ags typically induces Ag-specific systemic nonresponsiveness. However, we have found that feeding a model food protein, OVA, to helminth-infected mice primes for a systemic OVA-specific Th2 response. In this report we show that, in addition to creating a Th2-priming cytokine environment, helminth infection up-regulates costimulatory ... More
Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.
AuthorsKoo GC, Blake JT, Talento A, Nguyen M, Lin S, Sirotina A, Shah K, Mulvany K, Hora D, Cunningham P, Wunderler DL, McManus OB, Slaughter R, Bugianesi R, Felix J, Garcia M, Williamson J, Kaczorowski G, Sigal NH, Springer MS, Feeney W
JournalJ Immunol
PubMed ID9164927
'The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in ... More
Suppression of nonspecific binding of avidin-biotin complex (ABC) to proteins electroblotted to nitrocellulose paper.
AuthorsClark RK, Tani Y, Damjanov I
JournalJ Histochem Cytochem
PubMed ID3772081
'Nitrocellulose blots of cell extracts reacted in sequence with biotinylated lectins and horseradish peroxidase-labeled avidin-biotin complex (ABC) often show considerable nonspecific staining of protein bands. Experiments were performed to determine which of the components of the ABC were responsible for this and whether or not the nature and ionic strength ... More
Natural autoreactive B cells in transgenic mice reproduce an apparent paradox to the clonal tolerance theory.
'Naturally occurring autoreactive B cells are thought to be physically eliminated or rendered functionally silent through different mechanisms of tolerance. However, multireactive low affinity natural autoantibody-producing B cells seem to escape these mechanisms in normal adults and could constitute the B cell pool from which pathological autoantibodies can emerge. To ... More
Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.
AuthorsFerguson IA, Xian C, Barati E, Rush RA
JournalJ Neurosci Methods
PubMed ID11513942
'Established methods for monitoring regeneration of the corticospinal tract involve anterograde labelling of the cortical motor neuron. While wheat germ agglutinin-horseradish peroxidase conjugate has been used to anterogradely label these neurons, we demonstrate that this technique may not completely label the whole axon and fine terminal processes when this tracer ... More
Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function.
AuthorsNarravula S, Lennon PF, Mueller BU, Colgan SP
JournalJ Immunol
PubMed ID11046060
'During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5''-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5''-AMP. Extracellular 5''-AMP is metabolized to adenosine by surface-expressed 5''-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A(2B) receptors, ... More
Artifactual detection of biotin on histones by streptavidin.
AuthorsBailey LM, Ivanov RA, Wallace JC, Polyak SW,
JournalAnal Biochem
PubMed ID17920026
'Biotinylation is a recent addition to the list of reported posttranslational modifications made to histones. Holocarboxylase synthetase (HCS) and biotinidase have been implicated as biotinylating enzymes. However, the details of the mechanism and the regulation of biotin transfer on and off histones remains unclear. Here we report that in a ... More
Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin.
AuthorsHalfter W
JournalDev Biol
PubMed ID2433173
'Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra ... More
The N-terminal region and the mid-region complex of the integrin beta 2 subunit.
AuthorsTan SM, Robinson MK, Drbal K, van Kooyk Y, Shaw JM, Law SK
JournalJ Biol Chem
PubMed ID11477072
'In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of ... More
Nonsaturable binding indicates clustering of tau on the microtubule surface in a paired helical filament-like conformation.
AuthorsAckmann M, Wiech H, Mandelkow E
JournalJ Biol Chem
PubMed ID10869348
'Tau protein modulates microtubule dynamics and forms insoluble aggregates in Alzheimer''s disease. Because there is a discrepancy between reported affinities of Tau to microtubules, we determined the interaction over a wide concentration range using a sensitive enzyme-linked immunosorbent assay. We found that the interaction is biphasic and not monophasic as ... More
Transglutaminase activity in the eye: cross-linking in epithelia and connective tissue structures.
AuthorsRaghunath M, Cankay R, Kubitscheck U, Fauteck JD, Mayne R, Aeschlimann D, Schlötzer-Schrehardt U
JournalInvest Ophthalmol Vis Sci
PubMed ID10549636
'PURPOSE: To assess the distribution of transglutaminase (TGase) activity in ocular tissues and the target structures for cross-linking. METHODS: Cryosections from human and cynomolgus monkey eyes were incubated with the biotinylated amine donor substrate cadaverine (biotC), which was subsequently visualized with streptavidin-peroxidase. Confocal laser scanning was used to colocalize biotC ... More
Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes.
AuthorsGarza LA, Birnbaum MJ
JournalJ Biol Chem
PubMed ID10644714
'The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes ... More
The role of alpha and beta chains in ligand recognition by beta 7 integrins.
AuthorsHiggins JM, Cernadas M, Tan K, Irie A, Wang J, Takada Y, Brenner MB
JournalJ Biol Chem
PubMed ID10837471
'Integrins alpha(E)beta(7) and alpha(4)beta(7) are involved in localization of leukocytes at mucosal sites. Although both alpha(E)beta(7) and alpha(4)beta(7) utilize the beta(7) chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the ... More
The mechanisms of generation and propagation of synchronized bursting in developing networks of cortical neurons.
AuthorsMaeda E, Robinson HP, Kawana A
JournalJ Neurosci
PubMed ID7472441
'The characteristics and mechanisms of synchronized firing in developing networks of cultured cortical neurons were studied using multisite recording through planar electrode arrays (PEAs). With maturation of the network (from 3 to 40 d after plating), the frequency and propagation velocity of bursts increased markedly (approximately from 0.01 to 0.5 ... More
Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function.
AuthorsTan SM, Hyland RH, Al-Shamkhani A, Douglass WA, Shaw JM, Law SK
JournalJ Immunol
PubMed ID10946284
'LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 ... More
Patterning enzymes inside microfluidic channels via photoattachment chemistry.
AuthorsHolden MA, Jung SY, Cremer PS
JournalAnal Chem
PubMed ID15053641
'We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, ... More
Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays.
AuthorsBobrow MN, Harris TD, Shaughnessy KJ, Litt GJ
JournalJ Immunol Methods
PubMed ID2558138
'A novel signal amplification method, catalyzed reporter deposition (CARD), and its application to immunoassays is described. The method involves utilizing an analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter on the surface in a solid-phase immunoassay. In the examples described, deposition of reporter is facilitated by using ... More
Direct effects of radiation on the avidin-biotin system. Absence of energy transfer.
AuthorsKempner ES, Miller JH
JournalJ Biol Chem
PubMed ID2203786
Frozen solutions of biotinylated glucose-6-phosphate dehydrogenase and fluorescently tagged avidin were exposed to high energy ionizing radiation. Parallel experiments with peroxidase coupled to streptavidin and with biotinylated phycoerythrin were also performed. The loss of function of each compound was analyzed according to target theory. Target analysis revealed that the radiation-sensitive ... More
Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates.
AuthorsCole SR, Ashman LK, Ey PL
JournalMol Immunol
PubMed ID3309626
A method has been developed in which the conventional radioiodine label is replaced by non-radioactive biotin in studies involving the immunoprecipitation and analysis of cell surface antigens. The labelling reagent, d-biotinyl-N-hydroxysulfosuccinimide ester (NHSS-biotin), reacts preferentially with lysine residues in polypeptides and possibly also with free amino-groups on carbohydrates and lipids. ... More
An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2.
AuthorsRundle NT, Nelson J, Flory MR, Joseph J, Th'ng J, Aebersold R, Dasso M, Andersen RJ, Roberge M
JournalACS Chem Biol
PubMed ID17168522
Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit ... More
A gnotobiotic transgenic mouse model for studying interactions between small intestinal enterocytes and intraepithelial lymphocytes.
AuthorsMysorekar IU, Lorenz RG, Gordon JI,
JournalJ Biol Chem
PubMed ID12138109
The mouse intestinal epithelium undergoes continuous renewal throughout life. Intraepithelial lymphocytes (IELs) represent a significant fraction of this epithelium and play an important role in intestinal mucosal barrier function. We have generated a germ-free transgenic mouse model to examine the effects of a genetically engineered proliferative abnormality in the principal ... More
Robust fluorescent detection of protein fatty-acylation with chemical reporters.
AuthorsCharron G, Zhang MM, Yount JS, Wilson J, Raghavan AS, Shamir E, Hang HC,
JournalJ Am Chem Soc
PubMed ID19281244
Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ... More
Horseradish peroxidase: a modern view of a classic enzyme.
AuthorsVeitch NC,
JournalPhytochemistry
PubMed ID14751298
Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed ... More
Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice.
AuthorsKawasaki S, Takizawa H, Yoneyama H, Nakayama T, Fujisawa R, Izumizaki M, Imai T, Yoshie O, Homma I, Yamamoto K, Matsushima K
JournalJ Immunol
PubMed ID11160256
Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. ... More
Preparation of biotinylated, affinity-purified antibodies for enzyme-linked immunoassays using blotting membrane as an antigen support.
A method combining the affinity purification and biotinylation of antibodies has been developed utilizing the high-capacity binding of blotting membranes and glutaraldehyde treatment to immobilize antigen. Following reaction of the antisera with the membrane-bound antigen, the biotinylation of the antibodies is performed in situ, before release of the IgG-biotin complex ... More
Determination of biotin concentration by a competitive enzyme-linked immunosorbent assay (ELISA) method.
AuthorsChang YS, Wu CH, Chang RJ, Shiuan D
JournalJ Biochem Biophys Methods
PubMed ID7699208
A method based on competitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of biotin concentrations which takes advantage of the extraordinarily high affinity between biotin and avidin. The biotin assay consisted of two steps, (i) a competition reaction between excess streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and solutions of known ... More
Immunological capture of nucleic acid hybrids and application to nonradioactive DNA probe assay.
AuthorsRashtchian A, Eldredge J, Ottaviani M, Abbott M, Mock G, Lovern D, Klinger J, Parsons G
JournalClin Chem
PubMed ID2441901
Antibodies specific for DNA:RNA hybrids were coated onto polystyrene test tubes and applied to hybridization assays involving DNA and RNA. Synthetic DNA probes complementary to 16S rRNA of Campylobacter were labeled with biotin and hybridized to ribosomal RNA directly in lysates of bacterial cells. After hybridization, DNA:RNA hybrids were captured ... More
Large-scale screening assay to measure epidermal growth factor internalization.
Authorsde Wit R, Hendrix CM, Boonstra J, Verkleu AJ, Post JA
JournalJ Biomol Screen
PubMed ID10894756
Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the ... More
The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras.
AuthorsRizzo MA, Shome K, Watkins SC, Romero G
JournalJ Biol Chem
PubMed ID10801816
The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). ... More
ELISA-based assay for scatchard analysis of ligand-receptor interactions.
AuthorsVieira A
JournalMol Biotechnol
PubMed ID9951704
A simple, nonradioactive method is presented that can be used for performing large numbers of binding assays of cell membrane receptors with their ligands. The method adopts the simple membrane preparation and biotin-based quantitation methods of the semi-intact cell endocytosis assays. After binding of the biotinylated ligand to its receptors ... More
A method for selective intracellular labeling of immunostained neurons in turtle retina.
AuthorsFernandez E, Kolb H
JournalJ Histochem Cytochem
PubMed ID7680680
We describe a method for direct intracellular staining under visual control of immunolabeled neurons in the turtle retina. Substance P was the antiserum used. It labels two different sizes of ganglion cells in turtle retina. Intracellular labeling under visual control was achieved by iontophoresis of Lucifer yellow or Neurobiotin. The ... More
Improved cervical smear assessment using antibodies against proteins that regulate DNA replication.
AuthorsWilliams GH, Romanowski P, Morris L, Madine M, Mills AD, Stoeber K, Marr J, Laskey RA, Coleman N
JournalProc Natl Acad Sci U S A
PubMed ID9843993
Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate ... More
Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1.
AuthorsPetris MJ, Smith K, Lee J, Thiele DJ
JournalJ Biol Chem
PubMed ID12501239
Copper uptake at the plasma membrane and subsequent delivery to copper-dependent enzymes is essential for many cellular processes, including mitochondrial oxidative phosphorylation, free radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. However, intracellular levels of this nutrient must be controlled because it is potentially toxic in excess concentrations. The hCtr1 ... More
The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis.
AuthorsDanen EH, Sonneveld P, Brakebusch C, Fassler R, Sonnenberg A
JournalJ Cell Biol
PubMed ID12486108
We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at ... More
Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation.
AuthorsKelso RJ, Hudson AM, Cooley L
JournalJ Cell Biol
PubMed ID11854310
The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies ... More
A note on the use of biocytin in anterograde tracing studies in the central nervous system: application at both light and electron microscopic level.
AuthorsIzzo PN
JournalJ Neurosci Methods
PubMed ID1712060
A new neuroanatomical method based on the anterograde transport of biocytin has recently been reported. The validity of this method is examined closely on a number of well established pathways in the central nervous system, as well as the experimental parameters necessary for its effective use. iontophoretic application of this ... More
The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells.
AuthorsYoshimori T, Yamagata F, Yamamoto A, Mizushima N, Kabeya Y, Nara A, Miwako I, Ohashi M, Ohsumi M, Ohsumi Y
JournalMol Biol Cell
PubMed ID10679028
The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. ... More
The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts.
AuthorsMourton T, Hellberg CB, Burden-Gulley SM, Hinman J, Rhee A, Brady-Kalnay SM
JournalJ Biol Chem
PubMed ID11278757
PTPmu, an Ig superfamily receptor protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts with the cadherin-catenin complex. The signaling pathway downstream of PTPmu is unknown; therefore, we used a yeast two-hybrid screen to identify additional PTPmu interacting proteins. The membrane-proximal catalytic domain of PTPmu was used as bait. Sequencing of two ... More
Paxillin binding to a conserved sequence motif in the alpha 4 integrin cytoplasmic domain.
AuthorsLiu S, Ginsberg MH
JournalJ Biol Chem
PubMed ID10781578
alpha(4)beta(1) integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other beta(1) integrins. Paxillin, a signaling adapter protein, binds tightly to the alpha(4) cytoplasmic domain and is implicated in alpha(4) integrin signaling. We now report the mapping of a paxillin-binding site in ... More
Signaling through TNF receptor p55 in TNF-alpha-deficient mice alters the CXCL13/CCL19/CCL21 ratio in the spleen and induces maturation and migration of anergic B cells into the B cell follicle.
The organization of secondary lymphoid tissues into distinct T and B cell compartments supports proper regulation of an immune response to foreign Ags. In the splenic white pulp, this compartmentalization is also thought to be important in the maintenance of B cell tolerance. Using lymphotoxin-alpha-(LT-alpha)-, TNF-alpha-, or TNFRp55-deficient mice, all ... More
Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology.
AuthorsConboy MJ, Cyert MS
JournalMol Biol Cell
PubMed ID10888679
We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the ... More
The corticostriatal and corticotectal projections of the feline lateral suprasylvian cortex demonstrated with anterograde biocytin and retrograde fluorescent techniques.
AuthorsNorita M, McHaffie JG, Shimizu H, Stein BE
JournalNeurosci Res
PubMed ID1710043
The relationship between the visual cortex and the striatum (ST) of the cat is poorly understood. The present experiments were an attempt to determine if regions along the lateral suprasylvian cortex (LS), known to send dense visual projections to the superior colliculus (SC), also project to the striatum and, if ... More
The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.
Authorsvan der Loos CM, Göbel H
JournalJ Histochem Cytochem
PubMed ID10990496
The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal ... More
Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence.
AuthorsKane SA, Fleener CA, Zhang YS, Davis LJ, Musselman AL, Huang PS
JournalAnal Biochem
PubMed ID10640350
The p53 tumor suppressor protein is activated and stabilized in response to DNA damage, resulting in cell cycle arrest or apoptosis. HMD2 is a negative regulator of p53. Binding of p53 by HDM2 traffics p53 from the nucleus to the cytoplasm where it is recognized and targeted for ubiquitin-mediated degradation ... More
Control of intracellular movement of connexins by E-cadherin in murine skin papilloma cells.
AuthorsHernandez-Blazquez FJ, Joazeiro PP, Omori Y, Yamasaki H
JournalExp Cell Res
PubMed ID11640887
The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 ... More
A rapid chemiluminescent method for quantitation of human DNA.
AuthorsWalsh PS, Varlaro J, Reynolds R
JournalNucleic Acids Res
PubMed ID1408822
A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows ... More
Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation.
AuthorsRaaphorst FM, Otte AP, van Kemenade FJ, Blokzijl T, Fieret E, Hamer KM, Satijn DP, Meijer CJ
JournalJ Immunol
PubMed ID11342607
BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in ... More
15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit.
AuthorsCernuda-Morollón E, Pineda-Molina E, Cañada FJ, Pérez-Sala D
JournalJ Biol Chem
PubMed ID11466314
Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-kappaB. Here we explore the possibility that the NF-kappaB subunit p50 may be a target for the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This prostaglandin inhibited the DNA binding ability of recombinant p50 in ... More
Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures.
AuthorsKirkeby S, Bøg-Hansen TC, Moe D, Garbarsch C
JournalHistochem J
PubMed ID1717410
Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline ... More
Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates.
AuthorsMartin K, Hart C, Liu J, Leung WY, Patton WF
JournalProteomics
PubMed ID12872222
Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination ... More
CD45 function is regulated by an acidic 19-amino acid insert in domain II that serves as a binding and phosphoacceptor site for casein kinase 2.
AuthorsGreer SF, Wang Y, Raman C, Justement LB
JournalJ Immunol
PubMed ID11390469
In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B ... More
Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22.
AuthorsBobbitt KR, Justement LB
JournalJ Immunol
PubMed ID11067914
The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that ... More
Comparative morphology of three types of projection-identified pyramidal neurons in the superficial layers of cat visual cortex.
AuthorsMatsubara JA, Chase R, Thejomayen M
JournalJ Comp Neurol
PubMed ID8866848
The morphology and dendritic organization of corticocortical neurons in the superficial layers of area 18 that project to area 17 were studied by intracellular injection of lucifer yellow in the fixed-slice preparation. This corticocortical population contains primarily standard pyramidal cells, but occasional nonpyramidal, modified, fusiform, star, and inverted pyramidal cells ... More
Trafficking and proteolytic release of epidermal growth factor receptor ligands are modulated by their membrane-anchoring domains.
AuthorsDong J, Wiley HS
JournalJ Biol Chem
PubMed ID10617651
Ligands that bind to the epidermal growth factor (EGF) receptor are initially synthesized as integral membrane proteins that are released from the cell surface by regulated proteolysis. To study the role of the membrane-anchoring domain in ligand release, we made two artificial ligands. The first possessed the membrane-anchoring domain from ... More
The proamphiregulin cytoplasmic domain is required for basolateral sorting, but is not essential for constitutive or stimulus-induced processing in polarized Madin-Darby canine kidney cells.
AuthorsBrown CL, Coffey RJ, Dempsey PJ
JournalJ Biol Chem
PubMed ID11382759
In this study, the role of the amphiregulin precursor (pro-AR) cytoplasmic domain in the basolateral sorting and cell-surface processing of pro-AR in polarized epithelial cells has been investigated using Madin-Darby canine kidney cells stably expressing various human pro-AR forms. Our results demonstrate that newly synthesized wild-type pro-AR (50 kDa) is ... More