Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages.
AuthorsBenten WP,Lieberherr M,Stamm O,Wrehlke C,Guo Z,Wunderlich F
JournalMolecular biology of the cell
PubMed ID10512854
Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca(2+)], which is due to release of Ca(2+) from intracellular Ca(2+) stores. ... More
Abnormal regulation of retinoic acid receptor beta2 expression and compromised allograft rejection in transgenic mice expressing antisense sequences to retinoic acid receptor beta1 and beta3.
Transgenic mice carrying antisense sequences common to the retinoic acid receptors (RAR) beta1 and beta3 were produced to examine roles of RARbeta1 and beta3 in the immune system. There were no significant changes of endogenous RARbeta1/beta3 expression at the mRNA level in T cells, B cells, and macrophages of the ... More
Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: evidence of a cellular basis for the adipostat.
AuthorsCowley MA, Pronchuk N, Fan W, Dinulescu DM, Colmers WF, Cone RD
JournalNeuron
PubMed ID10677034
'Energy stores are held relatively constant in many mammals. The circuitry necessary for maintaining energy homeostasis should (1) sense the amount of energy stored in adipose tissue, (2) sense and integrate the multiple opposing signals regarding nutritional state, and (3) provide output regulating energy intake and expenditure to maintain energy ... 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
Galanin-5-hydroxytryptamine interactions: electrophysiological, immunohistochemical and in situ hybridization studies on rat dorsal raphe neurons with a note on galanin R1 and R2 receptors.
AuthorsXu ZQ, Zhang X, Pieribone VA, Grillner S, Hökfelt T
JournalNeuroscience
PubMed ID9722143
'Galaninergic mechanisms related to 5-hydroxytryptamine neurons in the dorsal raphe nucleus of the rat were analysed using electrophysiology, immunohistochemistry and in situ hybridization. Galanin caused a dose-dependent hyperpolarization accompanied by a decrease in membrane resistance in most 5-hydroxytryptamine-sensitive dorsal raphe neurons. The galanin-induced outward current reversed at about - 105 ... More
Tec kinase signaling in T cells is regulated by phosphatidylinositol 3-kinase and the Tec pleckstrin homology domain.
AuthorsYang WC, Ching KA, Tsoukas CD, Berg LJ
JournalJ Immunol
PubMed ID11123316
'Tec, the prototypical member of the Tec family of tyrosine kinases, is abundantly expressed in T cells and other hemopoietic cell types. Although the functions of Itk and Txk have recently been investigated, little is known about the role of Tec in T cells. Using antisense oligonucleotide treatment to deplete ... More
Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation.
AuthorsAdderley SR, Fitzgerald DJ
JournalJ Biol Chem
PubMed ID10681563
'The platelet integrin glycoprotein (GP) IIb/IIIa, which mediates platelet aggregation, has been the target for novel antiplatelet agents, the GPIIb/IIIa antagonists. Several GPIIb/IIIa antagonists have been developed based on the peptide RGDS present in adhesion proteins, including the principle ligand fibrinogen. The apoptosis enzyme, procaspase-3, contains an RGD-recognition sequence and ... More
The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain.
AuthorsGerrard SR, Mecklem AB, Stevens TH
JournalTraffic
PubMed ID11208059
'Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar ... More
Topologically restricted appearance in the developing chick retinotectal system of Bravo, a neural surface protein: experimental modulation by environmental cues.
Authorsde la Rosa EJ, Kayyem JF, Roman JM, Stierhof YD, Dreyer WJ, Schwarz U
JournalJ Cell Biol
PubMed ID2269667
'A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. ... More
Aberrant trafficking of the B cell receptor Ig-alpha beta subunit in a B lymphoma cell line.
AuthorsCondon C, Hourihane SL, Dang-Lawson M, Escribano J, Matsuuchi L
JournalJ Immunol
PubMed ID10903747
'The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the ... 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
Characterization of a CD43/leukosialin-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells.
'The monoclonal antibody (mAb) J393 induces apoptosis in Jurkat T-cells. NH2-terminal amino acid sequence analysis identified the 140-kDa surface antigen for mAb J393 as CD43/leukosialin, the major sialoglycoprotein of leukocytes. While Jurkat cells co-expressed two discrete cell-surface isoforms of CD43, recognized by mAb J393 and mAb G10-2, respectively, only J393/CD43 ... More
Trafficking of matrix collagens through bone-resorbing osteoclasts.
AuthorsNesbitt SA, Horton MA
JournalScience
PubMed ID9092478
'An intracellular pathway for proteins liberated from mineralized matrix during resorption was identified in osteoclasts. Analysis by confocal microscopy of sites of active bone resorption showed that released matrix proteins, including degraded type I collagen, were endocytosed along the ruffled border within the resorption compartment and transcytosed through the osteoclast ... More
Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-alpha.
AuthorsGarcía-Ruiz C, Colell A, Morales A, Calvo M, Enrich C, Fernández-Checa JC
JournalJ Biol Chem
PubMed ID12118012
'The interaction of mitochondria with proapoptotic proteins activates apoptosis pathways. Previous findings have identified ganglioside GD3 (GD3) as an emerging apoptotic lipid intermediate that targets mitochondria in response to death signals. Using immunoelectron and laser scanning confocal microscopy, we characterize the trafficking of GD3 to mitochondria in response to tumor ... More
Rac1 and Cdc42 are required for phagocytosis, but not NF-kappaB-dependent gene expression, in macrophages challenged with Pseudomonas aeruginosa.
AuthorsLee DJ, Cox D, Li J, Greenberg S
JournalJ Biol Chem
PubMed ID10617597
'Macrophages respond to Gram-negative bacterial pathogens by phagocytosis and pro-inflammatory gene expression. These responses may require GTPases that have been implicated in cytoskeletal alterations and activation of NF-kappaB. To determine the role of Rac1 and Cdc42 in signal transduction events triggered by Pseudomonas aeruginosa, we expressed GTP binding-deficient alleles of ... More
Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.
AuthorsLee JY, Qu-Petersen Z, Cao B, Kimura S, Jankowski R, Cummins J, Usas A, Gates C, Robbins P, Wernig A, Huard J
JournalJ Cell Biol
PubMed ID10973997
'Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that ... More
An adherent cell model to study different stages of apoptosis.
AuthorsDesjardins LM, MacManus JP
JournalExp Cell Res
PubMed ID7843282
'Apoptosis in the classical thymocyte model occurs very rapidly making it difficult to study the intermediate steps in the process. An alternative adherent cell model is characterized and proposed in this paper. HT29 cells treated with a teniposide were collected at various times for morphological and biochemical assessments. Large DNA ... More
Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system.
AuthorsShen Y, Mani S, Donovan SL, Schwob JE, Meiri KF
JournalJ Neurosci
PubMed ID11756507
'Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (-/-) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC) in vivo. Instead, ... More
Use of methacrylate De-embedding protocols for in situ hybridization on semithin plastic sections with multiple detection strategies.
AuthorsWarren KC, Coyne KJ, Waite JH, Cary SC
JournalJ Histochem Cytochem
PubMed ID9446821
'New plastic resins are gradually replacing traditional paraffin-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing ... More
Deletion analogues of transportan.
AuthorsSoomets U, Lindgren M, Gallet X, Hällbrink M, Elmquist A, Balaspiri L, Zorko M, Pooga M, Brasseur R, Langel U
JournalBiochim Biophys Acta
PubMed ID10930519
'Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular ... More
Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.
AuthorsLegrand C, Gilles C, Zahm JM, Polette M, Buisson AC, Kaplan H, Birembaut P, Tournier JM
JournalJ Cell Biol
PubMed ID10427102
'Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, ... More
Cell-adhesive properties of streptavidin are mediated by the exposure of an RGD-like RYD site.
AuthorsAlon R, Bayer EA, Wilchek M
JournalEur J Cell Biol
PubMed ID1425765
'The interaction of streptavidin with various cell systems was studied using fluorescent derivatives of the protein. The native unprocessed form of streptavidin bound to cells at low levels and in a nonspecific manner. In contrast, both the truncated "core" streptavidin (the commercially available form) and the biotin-blocked unprocessed protein bound ... More
Measurement of estrogen receptors in intact cells by flow cytometry.
AuthorsCao S, Hudnall SD, Kohen F, Lu LJ
JournalCytometry
PubMed ID11002266
'BACKGROUND: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. ... More
The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and beta1-integrins.
AuthorsArregui C, Pathre P, Lilien J, Balsamo J
JournalJ Cell Biol
PubMed ID10851023
'Cadherins and integrins must function in a coordinated manner to effectively mediate the cellular interactions essential for development. We hypothesized that exchange of proteins associated with their cytoplasmic domains may play a role in coordinating function. To test this idea, we used Trojan peptides to introduce into cells and tissues ... 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
Cocaine- and amphetamine-regulated transcript in the rat vagus nerve: A putative mediator of cholecystokinin-induced satiety.
AuthorsBroberger C, Holmberg K, Kuhar MJ, Hökfelt T
JournalProc Natl Acad Sci U S A
PubMed ID10557351
'Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the central nervous system. Recent studies have pointed to a role for CART-derived peptides in inhibiting feeding behavior. Although these actions have generally been attributed to hypothalamic CART, it remains to be determined whether additional CART pathways exist that link signals ... More
Chemical chaperones enhance superantigen and conventional antigen presentation by HLA-DM-deficient as well as HLA-DM-sufficient antigen-presenting cells and enhance IgG2a production in vivo.
AuthorsGhumman B, Bertram EM, Watts TH
JournalJ Immunol
PubMed ID9759841
'Chemical chaperones, first defined in studies of mutant cystic fibrosis transmembrane conductance regulator proteins, are small molecules that act as stabilizers of proteins in their native state and have the ability in some cases to rescue protein-folding mutants within cells. HLA-DM is an MHC II-specific molecular chaperone that facilitates peptide ... More
Lipid-modified, cysteinyl-containing peptides of diverse structures are efficiently S-acylated at the plasma membrane of mammalian cells.
AuthorsSchroeder H, Leventis R, Shahinian S, Walton PA, Silvius JR
JournalJ Cell Biol
PubMed ID8707845
'A variety of cysteine-containing, lipid-modified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid-modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S-acylation of plasma membrane-associated ... More
Two separate signals act independently to localize a yeast late Golgi membrane protein through a combination of retrieval and retention.
AuthorsBryant NJ, Stevens TH
JournalJ Cell Biol
PubMed ID9015300
'The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization ... More
NK cell trafficking and cytokine expression in splenic compartments after IFN induction and viral infection.
AuthorsSalazar-Mather TP, Ishikawa R, Biron CA
JournalJ Immunol
PubMed ID8816415
'Studies were undertaken to characterize mechanisms for NK cell cytokine delivery in vivo. Conditions of systemic IFN-beta expression elicited by polyinosinic-polycytidylic acid (poly(I:C)) treatment or IFN-alpha beta production during lymphocytic choriomeningitis virus or murine cytomegalovirus infections resulted in profound splenic histologic changes, with relocalization of nucleated cells from red to ... More
T cells from Jak3-deficient mice have intact TCR signaling, but increased apoptosis.
AuthorsThomis DC, Lee W, Berg LJ
JournalJ Immunol
PubMed ID9366394
'The Jak family tyrosine kinase, Jak3, is involved in signaling through cytokine receptors utilizing the common gamma-chain (gamma(c)). Mice and humans lacking Jak3 have severe immune deficiencies, including defects in B and T lymphocyte development and function. In particular, Jak3-deficient mice have mature T cells with an activated phenotype, yet ... More
Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells.
AuthorsGreen SA, Kelly RB
JournalJ Cell Biol
PubMed ID1313438
'Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a ... More
Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects.
AuthorsOmori Y, Mesnil M, Yamasaki H
JournalMol Biol Cell
PubMed ID8816997
'We have characterized the function of connexin (Cx) 32 gene mutations found in X-linked dominant Charcot-Marie-Tooth disease with respect to their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant negative mechanism. We prepared four types of Cx32 mutant cDNAs and transfected them ... More
Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts.
AuthorsAzuma Y, Kaji K, Katogi R, Takeshita S, Kudo A
JournalJ Biol Chem
PubMed ID10671521
'Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the ... More
Subtypes Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in pro-opiomelanocortin- and neuropeptide-Y-containing neurons of the rat hypothalamic arcuate nucleus.
AuthorsBroberger C, Landry M, Wong H, Walsh JN, Hökfelt T
JournalNeuroendocrinology
PubMed ID9430445
'The arcuate nucleus of the hypothalamus houses a number of neurochemically different cell populations. Among these, a dense cluster of small neuropeptide-Y (NPY)-expressing neurons is located in its ventromedial subdivision and a pro-opiomelanocortin (POMC)-expressing neuron population in its ventrolateral part. Furthermore, both neuropeptide Y Y1 and Y2 receptors (Y1-Rs and ... More
Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways.
AuthorsVallejo AN, Schirmer M, Weyand CM, Goronzy JJ
JournalJ Immunol
PubMed ID11086066
'CD4(+)CD28(null) T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases. Contrary to paradigm, they are functionally active and persist over many years. Such clonogenic potential and longevity suggest altered ... More
Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.
'After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5- (and -6)-carboxy-x-rhodamine], was microinjected into living oocytes. Biotinylated ... 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
Nonproliferating bystander CD4+ T cells lacking activation markers support HIV replication during immune activation.
AuthorsScales D, Ni H, Shaheen F, Capodici J, Cannon G, Weissman D
JournalJ Immunol
PubMed ID11342670
'HIV replicates primarily in lymphoid tissue and immune activation is a major stimulus in vivo. To determine the cells responsible for HIV replication during Ag-driven T cell activation, we used a novel in vitro model employing dendritic cell presentation of superantigen to CD4(+) T cells. Dendritic cells and CD4(+) T ... 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
Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
'Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I+/CD11b+/CD13+/CD34+/CD45RO+/MHC class II+/CD86+) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation ... More
Green- and red-fluorescent nanospheres for the detection of cell surface receptors by flow cytometry.
AuthorsBhalgat MK, Haugland RP, Pollack JS, Swan S, Haugland RP
JournalJ Immunol Methods
PubMed ID9831388
'Fluorescent probes serve as sensitive tools for obtaining structural and functional information in cellular systems. In spite of the high sensitivity provided by fluorescent reagents, cell surface receptors expressed in low numbers often escape detection with commonly used fluorescent probes. R-Phycoerythrin (R-PE), a molecule with a very high quantum yield, ... More
High gradient magnetic cell separation with MACS.
AuthorsMiltenyi S, Müller W, Weichel W, Radbruch A
JournalCytometry
PubMed ID1690625
'A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled ... More
Dynamic localization of CLIP-170 to microtubule plus ends is coupled to microtubule assembly.
AuthorsDiamantopoulos GS, Perez F, Goodson HV, Batelier G, Melki R, Kreis TE, Rickard JE
JournalJ Cell Biol
PubMed ID9885247
'CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends ... More
Purification and characterization of membrane proteins.
AuthorsKraehenbuhl JP, Bonnard C
JournalMethods Enzymol
PubMed ID2167430
Phospholipids undergo hop diffusion in compartmentalized cell membrane.
AuthorsFujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A
JournalJ Cell Biol
PubMed ID12058021
'The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at ... More
Estrogen suppresses mu-opioid- and GABAB-mediated hyperpolarization of hypothalamic arcuate neurons.
AuthorsKelly MJ, Loose MD, Ronnekleiv OK
JournalJ Neurosci
PubMed ID1319480
'The effects of estrogen on the response of hypothalamic arcuate neurons to mu-opioid and GABAB agonists were investigated. Intracellular recordings were made from arcuate neurons in slices prepared from ovariectomized guinea pigs that were pretreated with estrogen or vehicle. Estrogen shifted the dose-response curve to the mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) ... More
Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186.
AuthorsPalmer M, Jursch R, Weller U, Valeva A, Hilgert K, Kehoe M, Bhakdi S
JournalJ Biol Chem
PubMed ID8505320
'Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully ... More
Continuous photobleaching in vesicles and living cells: a measure of diffusion and compartmentation.
AuthorsDelon A, Usson Y, Derouard J, Biben T, Souchier C
JournalBiophys J
PubMed ID16428281
'We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t<0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t>0.1-5 s), for which ... More
A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment.
AuthorsRogalski AA
JournalJ Cell Biol
PubMed ID3114268
'An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., ... More
Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry.
AuthorsShattil SJ, Cunningham M, Hoxie JA
JournalBlood
PubMed ID3297204
'Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an ... More
Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles.
AuthorsLian W, Litherland SA, Badrane H, Tan W, Wu D, Baker HV, Gulig PA, Lim DV, Jin S
JournalAnal Biochem
PubMed ID15464962
'Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2''2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix ... More
Dual enhancement of triple immunofluorescence using two antibodies from the same species.
AuthorsNakamura A, Uchihara T
JournalJ Neurosci Methods
PubMed ID15020090
'Triple immunofluorescence method with two mouse monoclonal antibodies and another rabbit polyclonal antibody was established with catalyzed reporter deposition (CARD) amplification on thick floating sections from the rat cerebellum. One of the monoclonal antibodies (anti-calbindin), diluted maximally, probed with anti-mouse IgG-horseradish peroxidase (HRP) and amplified with Cy5-conjugated tyramide, immunolabeled cerebellar ... More
Optical imaging techniques (histochemical, immunohistochemical, and in situ hybridization staining methods) to visualize mitochondria.
AuthorsTanji K, Bonilla E
JournalMethods Cell Biol
PubMed ID11381601
Tyramide amplification allows anterograde tracing by horseradish peroxidase-conjugated lectins in conjunction with simultaneous immunohistochemistry
AuthorsKressel M
JournalJ Histochem Cytochem
PubMed ID9524199
Antibody-antigen binding constants determined in solution-phase with the threshold membrane-capture system: binding constants for anti-fluorescein, anti-saxitoxin, and anti-ricin antibodies.
AuthorsDill K, Lin M, Poteras C, Fraser C, Hafeman DG, Owicki JC, Olson JD
JournalAnal Biochem
PubMed ID8203727
Affinities of various monoclonal and polyclonal antibodies for fluorescein-containing antigens, saxitoxin and ricin, were determined by using a light addressable potentiometric sensor-based system (Threshold). The dissociation constants, determined from Scatchard plots, ranged from 2 x 10(-7) to approximately 3 x 10(-12) M. Dissociation constants for fluorescein and saxitoxin were compared ... More
Rapid production of quasi-stable antibody-phycoerythrin conjugates for use in flow cytometry.
AuthorsDale GL
JournalCytometry
PubMed ID9845444
A simple system is described for producing quasi-stable complexes between biotinylated antibodies and fluorochrome-labeled streptavidin. Optimal complexes were produced by mixing biotinylated antibody and phycoerythrin-streptavidin together at a molar ratio of 1:1 for 2 min, followed by the addition of soluble biotin to halt oligomerization. These complexes, which are stable ... More
Detection of DNA damage and identification of UV-induced photoproducts using the CometAssay kit.
AuthorsLemay M, Wood KA
JournalBiotechniques
PubMed ID10524327
We introduce the first commercially available comet assay for the detection and quantification of DNA damage in individual eukaryotic cells. The major difficulty of the comet assay is the preparation of the slides needed to immobilize the samples throughout the lysis and electrophoretic procedures. The CometAssay kit uses a proprietary ... More
Biotinylation: a simple method for labelling complement component C8 with preservation of functional activity.
AuthorsBhakdi S, Roth M, Hugo F
JournalJ Immunol Methods
PubMed ID2754261
Biotinylation of human C8 with the water-soluble biotin derivative biotinylamidohexanoic acid, N-hydroxysulfosuccinimide ester is an excellent method for labelling this terminal complement component with preservation of its functional activity. The biotinylated product can be detected both in native form and also following its incorporation into the terminal complement complexes. Detection ... More
Removal of osteoclast bone resorption products by transcytosis.
AuthorsSalo J, Lehenkari P, Mulari M, Metsikkö K, Väänänen HK
JournalScience
PubMed ID9092479
Osteoclasts are multinucleated cells responsible for bone resorption. During the resorption cycle, osteoclasts undergo dramatic changes in their polarity, and resorbing cells reveal four functionally and structurally different membrane domains. Bone degradation products, both organic and inorganic, were endocytosed from the ruffled border membrane. They were then found to be ... More
Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules.
AuthorsDarst SA, Ahlers M, Meller PH, Kubalek EW, Blankenburg R, Ribi HO, Ringsdorf H, Kornberg RD
JournalBiophys J
PubMed ID1901232
Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free ... More
Parallel microchannel-based measurements of individual erythrocyte areas and volumes.
AuthorsGifford SC, Frank MG, Derganc J, Gabel C, Austin RH, Yoshida T, Bitensky MW
JournalBiophys J
PubMed ID12524315
We describe a microchannel device which utilizes a novel approach to obtain area and volume measurements on many individual red blood cells. Red cells are aspirated into the microchannels much as a single red blood cell is aspirated into a micropipette. Inasmuch as there are thousands of identical microchannels with ... More
A profound deficiency in thymic progenitor cells in mice lacking Jak3.
AuthorsBaird AM, Lucas JA, Berg LJ
JournalJ Immunol
PubMed ID11034372
Humans and mice with genetic deficiencies that lead to loss of signaling through common gamma-chain (gammac)-containing cytokine receptors have severe defects in B and T lymphocytes. In humans, these deficiencies lead to a complete absence of T cells, whereas in mice, small thymuses give rise to normal numbers of peripheral ... More
Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.
AuthorsMurphy AM, Montell DJ
JournalJ Cell Biol
PubMed ID8636236
The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse ... More
Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axon mapping in vivo.
Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found throughout the nervous system. In the visual retinotectal projection, ELF-1, a ligand in the tectum, and its receptors in the retina show complementary gradients in expression and binding, indicating they may be ... More
Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces.
AuthorsLösche M, Piepenstock M, Diederich A, Grünewald T, Kjaer K, Vaknin D
JournalBiophys J
PubMed ID8298041
The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both in subphase water molecules and in the alkyl chains of the lipid ... More
7-amino-4-methylcoumarin-3-acetic acid-conjugated streptavidin permits simultaneous flow cytometry analysis of either three cell surface antigens or one cell surface antigen as a function of RNA and DNA content.
AuthorsAubry JP, Durand I, De Paoli P, Banchereau J
JournalJ Immunol Methods
PubMed ID1691233
We have investigated the use of the new coumarin dye AMCA (7-amino-4-methylcoumarin-3-acetic acid), as a fluorochrome for multiple color fluorescence analysis by flow cytometry. AMCA is now commercially available as a streptavidin conjugate. Its excitation wavelength is optimal at 351/364 nm with a UV argon ion laser and it emits ... More
The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.
AuthorsNastuk MA, Lieth E, Ma JY, Cardasis CA, Moynihan EB, McKechnie BA, Fallon JR
JournalNeuron
PubMed ID1660286
Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires ... More
Charge-coupled device camera-based detection of fluorescence-labeled proteins immobilized on nitrocellulose membranes.
AuthorsGraf R, Friedl P
JournalElectrophoresis
PubMed ID11332757
Proteins dotted on nitrocellulose membranes are biotinylated by reaction with a biotinyl-succinimide ester. The resulting biotinyl residues serve as specific binding sites for a subsequent streptavidin-based detection system. Using streptavidin-peroxidase, the proteins are visualized either by deposition of a colored formazan dye or by enhanced chemiluminescence the latter being twofold ... More
Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy.
AuthorsBenchaib M, Delorme R, Pluvinage M, Bryon PA, Souchier C
JournalHistochem Cell Biol
PubMed ID8877388
Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by ... More
VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment.
AuthorsGerrard SR, Bryant NJ, Stevens TH
JournalMol Biol Cell
PubMed ID10679018
Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a ... More
Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells.
AuthorsHayden GE, Walker KZ, Miller JF, Wotherspoon JS, Raison RL
JournalCytometry
PubMed ID2842118
A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody ... More
An effective and rapid method for functional characterization of immunoadsorbents using POROS beads and flow cytometry.
AuthorsAnderson NL, Haines LR, Pearson TW
JournalJ Proteome Res
PubMed ID15113098
To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were ... More
Use of random primer extension for concurrent amplification and nonradioactive labeling of nucleic acids.
AuthorsMackey J, Darfler M, Nisson P, Rashtchian A
JournalAnal Biochem
PubMed ID8214584
A method for efficient nonradioactive labeling of DNA with biotin using random primer extension has been developed. Under the conditions described, a significant amount of DNA synthesis occurs during incorporation of the nonradioactive label, resulting in amplification of the original template DNA. The effect of primer size, substrate concentration, enzyme ... More
Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy.
AuthorsWang G, Achim CL, Hamilton RL, Wiley CA, Soontornniyomkij V
JournalMethods
PubMed ID10491275
The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in ... More
Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization.
AuthorsBauman JG, Bentvelzen P
JournalCytometry
PubMed ID3208617
A method using flow cytometry and fluorescent in situ hybridization (ISH) to detect RNA in cells is described. L1210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single-stranded sense and antisense ... 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
Long-term culture of purified postnatal oligodendrocyte precursor cells. Evidence for an intrinsic maturation program that plays out over months.
AuthorsTang DG, Tokumoto YM, Raff MC
JournalJ Cell Biol
PubMed ID10704447
Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS). They develop from precursor cells (OPCs), some of which persist in the adult CNS. Adult OPCs differ in many of their properties from OPCs in the developing CNS. In this study we have purified OPCs from postnatal rat optic nerve ... More
Induction of germinal center B cell markers in vitro by activated CD4+ T lymphocytes: the role of CD40 ligand, soluble factors, and B cell antigen receptor cross-linking.
AuthorsLahvis GP, Cerny J
JournalJ Immunol
PubMed ID9257841
Following primary immunization, B cells differentiate to memory cells with help from T cells. The specialized path to B cell memory takes place in lymphoid germinal centers (GC), where mouse B cells up-regulate peanut agglutinin receptor (PNA-R), B7-2 (CD86), and MHC class II expression. Using an in vitro culture system, ... More
A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts.
AuthorsArora PD, Manolson MF, Downey GP, Sodek J, McCulloch CA
JournalJ Biol Chem
PubMed ID10945978
Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous alpha(2)beta(1) integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen ... More
Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide.
AuthorsJaye DL, Edens HA, Mazzucchelli L, Parkos CA
JournalJ Immunol
PubMed ID11390474
Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in ... More
Coupling of biotin-(poly(ethylene glycol))amine to poly(D,L-lactide-co-glycolide) nanoparticles for versatile surface modification.
AuthorsWeiss B, Schneider M, Muys L, Taetz S, Neumann D, Schaefer UF, Lehr CM
JournalBioconjug Chem
PubMed ID17590034
Generally, polymeric nanoparticles (NP) for drug targeting are designed to entrap the drug moiety in the core and to present the targeting moiety on the surface. However, in most cases, common preparation techniques of polymeric NP need to be specifically arranged for each compound to be entrapped or attached. In ... More
Synthesis of the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) and its use with the IAsys biosensor to study receptor-ligand interactions on model membranes.
AuthorsAltin JG, White FA, Easton CJ
JournalBiochim Biophys Acta
PubMed ID11470085
This work describes the synthesis and use of the chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). This lipid is readily dispersed in aqueous media, both alone and when mixed with carrier lipids like dimyristoylphosphatidylcholine (DMPC). Fluorescence microscopic examination of membranes deposited from NTA-DTDA-containing liposomes shows that NTA-DTDA mixes uniformly with the ... More
Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms.
AuthorsWallner G, Amann R, Beisker W
JournalCytometry
PubMed ID7679962
A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) ... More
Fascin is involved in the antigen presentation activity of mature dendritic cells.
AuthorsAl-Alwan MM, Rowden G, Lee TD, West KA
JournalJ Immunol
PubMed ID11123310
Maturation of dendritic cells (DC) is critical to their development into potent APCs. Upon maturation, DC up-regulate the expression of MHC class II as well as costimulatory and adhesion molecules, all of which are important in Ag presentation. In addition, they undergo structural changes characterized by the expression of numerous ... More
Ultrastructural localization of beta-actin and amphoterin mRNA in cultured cells: application of tyramide signal amplification and comparison of detection methods.
AuthorsPunnonen EL, Fages C, Wartiovaara J, Rauvala H
JournalJ Histochem Cytochem
PubMed ID9857217
We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding beta-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold ... More
Quantifying apoptosis in banked human brains using flow cytometry.
AuthorsOlano JP, Wolf D, Keherly M, Gelman BB
JournalJ Neuropathol Exp Neurol
PubMed ID8939200
Fragmentation of genomic DNA, a major biochemical feature of programmed cell death (apoptosis), is easily detected when apoptosis is prevalent. In brain tissue apoptotic cells are usually scarce and detection requires more sensitive techniques. We describe a highly sensitive method to quantify apoptosis in frozen human brain tissue using flow ... 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
The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts.
AuthorsLeno GH, Laskey RA
JournalJ Cell Biol
PubMed ID1993731
We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared ... More
Isolation and functional characterization of murine prostate stem cells.
AuthorsLawson DA, Xin L, Lukacs RU, Cheng D, Witte ON
JournalProc Natl Acad Sci U S A
PubMed ID17185413
The ability to isolate prostate stem cells is essential to explore their role in prostate development and disease. In vitro prostate colony- and sphere-forming assays were used to quantitatively measure murine prostate stem/progenitor cell enrichment and self-renewal. Cell surface markers were screened for their ability to positively or negatively enrich ... More
Agrin binds to the nerve-muscle basal lamina via laminin.
AuthorsDenzer AJ, Brandenberger R, Gesemann M, Chiquet M, Ruegg MA
JournalJ Cell Biol
PubMed ID9151673
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from ... More
Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy.
AuthorsBauman JG, Bayer JA, van Dekken H
JournalJ Microsc
PubMed ID1688948
A fluorescent in-situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single-cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3-D morphology perfectly preserved. The spatial distribution of ... More
RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts.
AuthorsKomarova SV, Pilkington MF, Weidema AF, Dixon SJ, Sims SM
JournalJ Biol Chem
PubMed ID12496256
RANK ligand (RANKL) induces activation of NFkappaB, enhancing the formation, resorptive activity, and survival of osteoclasts. Ca(2+) transduces many signaling events, however, it is not known whether the actions of RANKL involve Ca(2+) signaling. We investigated the effects of RANKL on rat osteoclasts using microspectrofluorimetry and patch clamp. RANKL induced ... More
The level of Z-DNA in metabolically active, permeabilized mammalian cell nuclei is regulated by torsional strain.
AuthorsWittig B, Dorbic T, Rich A
JournalJ Cell Biol
PubMed ID2921282
Permeabilized nuclei from mammalian cells encapsulated within agarose microbeads in an isotonic buffer are active in transcription and replication (Jackson, D. A., and P. R. Cook. 1985. EMBO (Eur. Mol. Biol. Organ.) J. 4:913-918). Their DNA is intact and the nuclei are accessible to macromolecules. Myeloma nuclei prepared in this ... More
Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases.
AuthorsHolinger EP, Chittenden T, Lutz RJ
JournalJ Biol Chem
PubMed ID10224090
The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce ... More
Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses.
AuthorsCaldwell JH, Schaller KL, Lasher RS, Peles E, Levinson SR
JournalProc Natl Acad Sci U S A
PubMed ID10779552
Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform ... More
Inhibition of endothelial vascular cell adhesion molecule-1 expression by nitric oxide involves the induction and nuclear translocation of IkappaBalpha.
AuthorsSpiecker M, Peng HB, Liao JK
JournalJ Biol Chem
PubMed ID9388244
The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-alpha requires the activation of nuclear factor-kappaB (NF-kappaB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IkappaBalpha. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-kappaB activation. To ... 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
Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining.
AuthorsHunyady B, Krempels K, Harta G, Mezey E
JournalJ Histochem Cytochem
PubMed ID8985127
The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the ... More
Characterization of the human B cell RAG-associated gene, hBRAG, as a B cell receptor signal-enhancing glycoprotein dimer that associates with phosphorylated proteins in resting B cells.
AuthorsVerkoczy LK, Guinn B, Berinstein NL
JournalJ Biol Chem
PubMed ID10749872
Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG-associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane-integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated ... More