Parathyroid hormone controls the size of the intracellular Ca(2+) stores available to receptors linked to inositol trisphosphate formation.
AuthorsShort AD, Taylor CW
JournalJ Biol Chem
PubMed ID10636879
'In HEK 293 cells stably expressing type 1 parathyroid (PTH) receptors, PTH stimulated release of intracellular Ca(2+) stores in only 27% of cells, whereas 96% of cells responded to carbachol. However, in almost all cells PTH potentiated the response to carbachol by about 3-fold. Responses to carbachol did not desensitize, ... More
Neuropeptides exert direct effects on rat thymic epithelial cells in culture.
AuthorsHead GM, Mentlein R, von Patay B, Downing JE, Kendall MD
JournalDev Immunol
PubMed ID9716910
'To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue ... More
Oligosaccharide organization of the beta-subunits of pig kidney Na+/K+-ATPase.
AuthorsAmler E, Staffolani R, Baranska J, Obsil T, Urbanová P, Bertoli E, Mazzanti L
JournalPhysiol Res
PubMed ID9730046
'The distance between the beta-subunits of Na+/K+-ATPase isolated from pig dark red kidney medulla was determined by Förster energy transfer. First, oligosaccharides of the beta-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSH ... More
Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine.
AuthorsGeoghegan KF, Stroh JG
JournalBioconjug Chem
PubMed ID1325188
'The 2-amino alcohol structure -CH(NH2)CH(OH)- exists in proteins and peptides in N-terminal Ser or Thr and in hydroxylysine. Its very rapid oxidation by periodate at pH 7 generates an aldehyde in the peptide and is the first step in a method for site-directed labeling with biotin or a fluorescent reporter. ... More
Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions.
AuthorsBecker DL, Evans WH, Green CR, Warner A
JournalJ Cell Sci
PubMed ID7615666
'Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding ... More
Nucleophile labeling of cysteine and serine protease substrates.
AuthorsWang Y, Rasnick D, Klaus J, Payan D, Brömme D, Anderson DC
JournalJ Biol Chem
PubMed ID8910464
'Dipeptides containing fluorescein or biotin have been incorporated into proteolytic substrate cleavage products of bovine serum albumin generated by human cathepsin S or neutrophil elastase and into a fragment of the 31-kDa interleukin 1beta precursor by human interleukin 1beta-converting enzyme. Incorporation of the nucleophile is blocked by prior inhibition of ... More
Identification of a pore lining segment in gap junction hemichannels.
AuthorsZhou XW, Pfahnl A, Werner R, Hudder A, Llanes A, Luebke A, Dahl G
JournalBiophys J
PubMed ID9129799
The ability of certain connexins to form open hemichannels has been exploited to study the pore structure of gap junction (hemi)channels. Cysteine scanning mutagenesis was applied to cx46 and to a chimeric connexin, cx32E(1)43, which both form patent hemichannels when expressed in Xenopus oocytes. The thiol reagent maleimido-butyryl-biocytin was used ... More
Skin electroporation: rapid measurements of the transdermal voltage and flux of four fluorescent molecules show a transition to large fluxes near 50 V.
AuthorsChen T, Segall EM, Langer R, Weaver JC
JournalJ Pharm Sci
PubMed ID9811492
Large molecular fluxes and tight control are highly desired for transdermal drug delivery, which motivated this study of molecular transport due to high voltage pulsing. We used a flow-through sampling system (time resolution of approximately 14 s) to measure the response of human skin in vitro to a series of ... More
Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.
AuthorsHoffmann PR, deCathelineau AM, Ogden CA, Leverrier Y, Bratton DL, Daleke DL, Ridley AJ, Fadok VA, Henson PM
JournalJ Cell Biol
PubMed ID11706053
Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish ... More
Distribution of the monochlorobimane-glutathione conjugate between nucleus and cytosol in isolated hepatocytes.
AuthorsBriviba K, Fraser G, Sies H, Ketterer B
JournalBiochem J
PubMed ID8379916
The nuclear/cytoplasmic distributions of the GSH and N-acetylcysteine conjugates of monochlorobimane (MCB) have been studied in rat hepatocytes in primary culture after microinjection into the cytoplasm. The immediately occurring fluorescence (within 1-2 s) in the nucleus was much greater than that in the cytoplasm. Other water-soluble fluorescent markers such as ... More
Active propagation of somatic action potentials into neocortical pyramidal cell dendrites.
AuthorsStuart GJ, Sakmann B
JournalNature
PubMed ID8107777
The dendrites of neurons in the mammalian central nervous system have been considered as electrically passive structures which funnel synaptic potentials to the soma and axon initial segment, the site of action potential initiation. More recent studies, however, have shown that the dendrites of many neurons are not passive, but ... More
Electroporation loading and photoactivation of caged InsP3: tools to investigate the relation between cellular ATP release in response to intracellular InsP3 elevation.
AuthorsBraet K, Mabilde C, Cabooter L, Rapp G, Leybaert L
JournalJ Neurosci Methods
PubMed ID14687677
Photolytic liberation of InsP(3) in single cells triggers cell-to-cell propagating calcium changes that are communicated by a gap junctional and a paracrine purinergic pathway involving InsP(3)-triggered ATP release. We investigated the relation between the InsP(3) stimulus and the resulting ATP release in ECV304 cells using UV photolysis of caged compounds ... More
Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.
AuthorsXu C, Zipfel W, Shear JB, Williams RM, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID8855254
Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's ... More
Exciton coupled circular dichroic studies of self-assembled brevetoxin-porphyrin conjugates in lipid bilayers and polar solvents.
AuthorsMatile S, Berova N, Nakanishi K
JournalChem Biol
PubMed ID8807867
BACKGROUND: Brevetoxins, involved in the 'red tide' as well as shellfish poisoning, are known to bind to cell membranes and membrane proteins. Brevetoxin B (BTX-B) interacts specifically with neuronal sodium channels. We recently found that BTX also induces selective ion movements across lipid bilayers through transmembrane BTX self-assemblies. RESULTS: We ... More
Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.
A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively ... More
Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1.
AuthorsNielsen PA, Beahm DL, Giepmans BN, Baruch A, Hall JE, Kumar NM
JournalJ Biol Chem
PubMed ID12154091
A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.9 gene ... More
Dynamin-mediated internalization of caveolae.
AuthorsHenley JR, Krueger EW, Oswald BJ, McNiven MA
JournalJ Cell Biol
PubMed ID9531550
The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial ... More
Molecular probes for muscarinic receptors: functionalized congeners of selective muscarinic antagonists.
AuthorsJacobson KA, Fischer B, van Rhee AM
JournalLife Sci
PubMed ID10188781
The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of ... More
Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.
AuthorsWhitaker JE, Haugland RP, Moore PL, Hewitt PC, Reese M, Haugland RP
JournalAnal Biochem
PubMed ID1724118
Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and ... More
tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation.
AuthorsPeng J, Tan C, Roberts GJ, Nikolaeva O, Zhang Z, Lapolla SM, Primorac S, Andrews DW, Lin J
JournalJ Biol Chem
PubMed ID17005564
During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax ... More
Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer.
The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically ... More
Modulation of dye-coupling and proliferation in cultured rat thymic epithelium by factors involved in thymulin secretion.
AuthorsHead GM, Mentlein R, Kranz A, Downing JE, Kendall MD
JournalJ Anat
PubMed ID9418992
Cultures of rat thymic epithelium were used to measure the effect of thymulin secretagogues on dye-coupling and proliferation. Dye-coupling was assessed after the injection of lucifer yellow dextran which cannot permeate the connexin pore of gap junctions and the smaller, permeant cascade blue. In addition to gap junctional communication, larger ... More
Functional gap junctions in corneal fibroblasts and myofibroblasts.
AuthorsSpanakis SG, Petridou S, Masur SK
JournalInvest Ophthalmol Vis Sci
PubMed ID9660479
PURPOSE: Within the corneal stroma, keratocytes communicate through gap junctions. These plasma membrane channels, which connect the cytoplasm of adjacent cells, are composed of connexins. In a cell culture model, an investigation was conducted to determine whether connexin-based gap junction intercellular communication is present in fibroblasts and myofibroblasts, both of ... More
Identification of microvascular transport pathways in skeletal muscle.
AuthorsWolf MB
JournalAm J Physiol
PubMed ID8048604
The solvent-drag reflection coefficient (sigma f) was measured from plasma disappearance (integral-mass balance method) for native albumin and four fluorescent solutes of radii from 2 to 16 nm in the isolated, plasma-perfused cat hindlimb preparation. The data for the smallest solutes were measured > 2 h after tracer addition and ... More