Enumeration of antigen-specific IgE responses at the single-cell level by an ELISA plaque assay.
AuthorsChen SS
JournalJ Immunol Methods
PubMed ID1703186
In vitro elicitation and enumeration of antigen-specific IgE-secreting cells in antigen-primed murine lymphocyte cultures can be reproducibly and accurately quantitated. Rat anti-mouse IgE monoclonal antibodies (Mab anti-epsilon) were developed. Antigen-specific IgE-secreting cells can be detected on antigen or MAb anti-epsilon coated nitrocellulose discs pasted to 24-well culture plates. This IgE ... More
Detection of low copy numbers of HPV DNA by fluorescent in situ hybridization combined with confocal microscopy as an alternative to in situ polymerase chain reaction.
AuthorsLizard G, Chignol MC, Souchier C, Roignot P, Chardonnet Y, Schmitt D
JournalJ Virol Methods
PubMed ID9672129
'In genital lesions infected by human papillomavirus (HPV), histological criteria and HPV DNA typing are of prognostic value. Therefore, non-radioactive methods such as in situ hybridization are used extensively since they preserve the histological organization of the tissue, and allow the detection and characterization of HPV DNA. However, the sensitivity ... 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
Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.
AuthorsOliver C, Sahara N, Kitani S, Robbins AR, Mertz LM, Siraganian RP
JournalJ Cell Biol
PubMed ID1370498
'The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 ... More
Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies.
AuthorsLongva KE, Blystad FD, Stang E, Larsen AM, Johannessen LE, Madshus IH
JournalJ Cell Biol
PubMed ID11864992
'EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl ... More
Simultaneous visualization of G- and F-actin in endothelial cells.
AuthorsHaugland RP, You W, Paragas VB, Wells KS, DuBose DA
JournalJ Histochem Cytochem
PubMed ID8308251
'We developed site-specific fluorescent probes that permit simultaneous microscopic observation of G- and F-actin in bovine endothelial cells. G-actin distribution was visualized with fluorescein-deoxyribonuclease I (DNAse I). F-actin was labeled with phalloidin conjugated to the new long-wavelength fluorophore BODIPY 581/591 (581-nm excitation, 591-nm emission), which is spectrally similar to Texas ... More
Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells.
AuthorsYaroslavskiy B, Watkins S, Donnenberg AD, Patton TJ, Steinman RA
JournalBlood
PubMed ID10216085
'A central question in hematopoiesis is how cell-cycling behavior changes during the emergence of the differentiated state. To further understand what genetic regulators might couple proliferation status to differentiation, we studied the expression of the cell-cycle inhibitors p21 and p27 during the in vitro differentiation of normal CD34(+) blast cells ... More
Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.
AuthorsBreininger JF, Baskin DG
JournalJ Histochem Cytochem
PubMed ID11101627
'To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, ... More
Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays.
AuthorsChandler DP, Newton GJ, Small JA, Daly DS
JournalAppl Environ Microbiol
PubMed ID12732571
'A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance ... More
AuthorsMendoza LG, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers M
JournalBiotechniques
PubMed ID10524321
'A new generation biochip is described as capable of supporting high-throughput (HT), multiplexed enzyme-linked immunosorbent assays (ELISAs). These biochips consist of an optically flat, glass plate containing 96 wells formed by an enclosing hydrophobic Teflon mask. The footprint dimensions of each well and the plate precisely match those of a ... 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
Biocytin: a versatile anterograde neuroanatomical tract-tracing alternative.
AuthorsKing MA, Louis PM, Hunter BE, Walker DW
JournalBrain Res
PubMed ID2479450
'Biocytin, a naturally occurring low molecular weight analog of biotin, was evaluated as a neuroanatomical tract-tracing marker in the adult rat brain. Since it retains high-affinity binding to avidin, biocytin can be labelled with avidinylated visualization reagents. Iontophoretic or pressure injections resulted in filling of cell bodies and dendrites around ... More
Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.
AuthorsGebeyehu G, Rao PY, SooChan P, Simms DA, Klevan L
JournalNucleic Acids Res
PubMed ID3588302
'A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated ... More
Cutting edge: germinal centers can be induced in the absence of T cells.
AuthorsLentz VM, Manser T
JournalJ Immunol
PubMed ID11418626
'Immunization of mice containing mutations that inactivate the TCR Cbeta and Cdelta genes with the T cell-independent (TI) type 2 Ag (4-hydroxy-3-nitrophenyl)acetyl-Ficoll induces clusters of peanut agglutinin-binding B cells in the spleen. These clusters are histologically indistinguishable from germinal centers (GCs) typical of T cell-dependent immune responses. They are located ... More
Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase.
AuthorsKumar A, Tchen P, Roullet F, Cohen J
JournalAnal Biochem
PubMed ID2837921
Synthetic oligonucleotides were tailed at the 3' end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The ... More
Cutting edge: myeloid complement C3 enhances the humoral response to peripheral viral infection.
AuthorsVerschoor A, Brockman MA, Knipe DM, Carroll MC
JournalJ Immunol
PubMed ID11509581
HSV-1 is the causative agent of cutaneous lesions, commonly referred to as cold sores. Primary exposure to the virus ordinarily occurs through the periphery, in particular through abraded skin or mucosal membranes. Under certain circumstances (e.g., in neonatals or AIDS patients), the infection becomes disseminated, often with severe consequences. Spread ... More
Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.
AuthorsRodgers JT, Patel P, Hennes JL, Bolognia SL, Mascotti DP
JournalAnal Biochem
PubMed ID10625515
We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect ... More
A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate.
AuthorsSlaughter TF, Achyuthan KE, Lai TS, Greenberg CS
JournalAnal Biochem
PubMed ID1359806
Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing. We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N'-dimethylcasein. The substrate 5-(biotinamido)pentylamine is covalently incorporated into N,N'-dimethylcasein by transglutaminase in a calcium-dependent reaction. The biotinylated product ... More
Versican interacts with chemokines and modulates cellular responses.
AuthorsHirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M
JournalJ Biol Chem
PubMed ID11083865
We previously reported that versican, a large chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding was strongly inhibited by the chondroitinase digestion of versican or by the addition of soluble ... More
Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.
AuthorsObzansky DM, Rabin BR, Simons DM, Tseng SY, Severino DM, Eggelte H, Fisher M, Harbron S, Stout RW, Di Paolo MJ
JournalClin Chem
PubMed ID1893577
A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active ... 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
A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation.
AuthorsJenkins BJ, Le F, Gonda TJ
JournalJ Biol Chem
PubMed ID10085105
The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMRalpha) and a common signal-transducing beta-subunit (hbetac) that is shared with the interleukin-3 and -5 receptors. We have previously identified a constitutively active extracellular point mutant of hbetac, I374N, that can confer factor independence on ... More
An improved, luminescent europium-based stain for detection of electroblotted proteins on nitrocellulose or polyvinylidene difluoride membranes.
AuthorsKemper C, Berggren K, Diwu Z, Patton WF
JournalElectrophoresis
PubMed ID11332756
SYPRO Rose Plus protein blot stain is an improved europium-based metal chelate stain for the detection of proteins on nitrocellulose and poly(vinylidene difluoride) (PVDF) membranes. Staining is achieved without covalently modifying the proteins. The stain may be excited with a 254 nm (UV-C), 302 nm (UV-B), or 365 nm (UV-A) ... More
In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.
AuthorsLehtonen S, Ora A, Olkkonen VM, Geng L, Zerial M, Somlo S, Lehtonen E
JournalJ Biol Chem
PubMed ID10913159
We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein ... More
The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets.
AuthorsParagas VB, Kramer JA, Fox C, Haugland RP, Singer VL
JournalJ Microsc
PubMed ID12000550
We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright ... More
The rearranged V(H) domain of a physiologically selected anti-single-stranded DNA antibody as a precursor for formation of IgM and IgG antibodies to diverse antigens.
It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse ... More
Characterization and identification of proteinases and proteinase inhibitors synthesized by temporomandibular joint disc cells.
AuthorsKapila S, Lee C, Richards DW
JournalJ Dent Res
PubMed ID7629341
The adult mammalian temporomandibular joint (TM) disc is a fibrocartilaginous tissue that undergoes normal developmental remodeling, requiring removal of the existing extracellular matrix and its replacement by new matrix macromolecules. This remodeling is probably mediated by matrix-degrading enzymes, but to date none has been demonstrated in association with the TMJ ... More
Differential gene regulation in human versus rodent hepatocytes by peroxisome proliferator-activated receptor (PPAR) alpha. PPAR alpha fails to induce peroxisome proliferation-associated genes in human cells independently of the level of receptor expresson.
AuthorsLawrence JW, Li Y, Chen S, DeLuca JG, Berger JP, Umbenhauer DR, Moller DE, Zhou G
JournalJ Biol Chem
PubMed ID11418601
We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) alpha agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In ... More