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Biomedical subjects

Barbara Baird

Publications and source records attributed to Barbara Baird.

18 recordsLinked to original sources
PubMed2006

Functionalized surface arrays for spatial targeting of immune cell signaling.

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.

Microscopy, Confocal↗
PubMed2006

Coexisting domains in the plasma membranes of live cells characterized by spin-label ESR spectroscopy.

The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.

Animals↗
PubMed2006

Dinitrophenyl ligand substrates and their application to immunosensors.

We present an approach for the development of highly specific and sensitive antibody based biosensors by chemically tailoring the sensor surface with materials that control specific and nonspecific binding of biologically relevant molecules. As a model system we employed surface immobilized 2,4-dinitrophenyl (DNP)-ligands that bind specifically to anti-DNP antibodies. Self-assembling characteristics and minimization of the nonspecific interactions were used in the ligand design. The redox activity of the DNP-head group was used to calculate the surface density (coverage) of these assemblies using cyclic voltammetry. Quartz crystal microbalance (QCM) and impedance analysis were used to assess the ligand-antibody interaction and estimate the quantity of antibodies bound to the surface. The ligand surface density and the QCM data were useful in determining the sensitivity of our model system. A simple two-step kinetic model was shown to fit the experimental data.

Antibodies↗
PubMed2005

Transmembrane sequences are determinants of immunoreceptor signaling.

To investigate structural features critical for signal initiation by Ag-stimulated immunoreceptors, we constructed a series of single-chain chimeric receptors that incorporate extracellular human Fc epsilonRIalpha for IgE binding, a variable transmembrane (TM) segment, and the ITAM-containing cytoplasmic tail of the TCR zeta-chain. We find that functional responses mediated by these receptors are strongly dependent on their TM sequences, and these responses are highly correlated to cross-link-dependent association with detergent-resistant lipid rafts. For one chimera designated alpha Fzeta, mutation of a TM cysteine abolishes robust signaling and lipid raft association. In addition, TM disulfide-mediated oligomerization of another chimeric receptor, alpha zetazeta, enhances signaling. These results demonstrate an important role for TM segments in immunoreceptor signaling and a strong correspondence between strength of signaling and cross-link-dependent partitioning into ordered membrane domains.

Amino Acid Sequence↗
PubMed2005

Lipid segregation and IgE receptor signaling: a decade of progress.

Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment. Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation. These and other results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and other cell surface receptors.

Animals↗
PubMed2005

High spatial resolution observation of single-molecule dynamics in living cell membranes.

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.

Animals↗
PubMed2005

Oligo(ethylene glycol) containing polymer brushes as bioselective surfaces.

The nitroxide-mediated polymerization of styrenic monomers containing oligo(ethylene glycol) (OEGn) moieties was chosen for the preparation of biocompatible polymer brushes tethered to silicon oxide surfaces due to the broad range of monomer structures available and the use of a nonmetallic initiator. These surfaces were characterized by near-edge X-ray absorption fine structure and water contact angle measurements. The biocompatibility of these grown polymer brushes was studied and compared with deposited assemblies of surface-bound OEGn-terminated silanes with selected chain lengths. Grown polymer brushes with short OEGn side chains suppressed protein adsorption significantly more than the deposited assemblies of short OEGn chains, and this was attributed to higher surface coverage by the brushes. Cell adhesion studies confirmed that OEGn-containing polymer brushes are particularly effective in preventing nonspecific adhesion. Studies of protein adsorption and cell localization carried out with specific ligands on surfaces patterned demonstrated the potential of these surface-tethered polymer brushes for the formation of micro- and nanoscale devices.

Adsorption↗
PubMed2004

Reconstitution of regulated phosphorylation of FcepsilonRI by a lipid raft-excluded protein-tyrosine phosphatase.

To examine the exquisite regulation of IgE-FcepsilonRI tyrosine phosphorylation by Lyn kinase that is stimulated by antigen-mediated cross-linking, we utilized co-expression of FcepsilonRI and Lyn in Chinese hamster ovary cells, which results in high basal levels of Lyn kinase activity and spontaneous phosphorylation of FcepsilonRI. We found that co-expression of a lipid raft-excluded transmembrane tyrosine phosphatase, PTPalpha, suppresses Lyn kinase activity and markedly reduces the level of spontaneous phosphorylation of FcepsilonRI, while facilitating its antigen-stimulated phosphorylation. Other tyrosine phosphatases, including SHP-1, CD45, and a lipid raft-preferring chimeric version of PTPalpha fail to reconstitute antigen-dependent FcepsilonRI phosphorylation. We concluded that both substrate specificity and submembrane location are critical to phosphatase-mediated regulation of Lyn kinase activity that supports activation of FcepsilonRI.

Animals↗
PubMed2004

In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells.

A direct degranulation assay has been developed to enable the use of RBL mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation. Release of beta-hexosaminidase into the extracellular milleu is widely used to characterize cellular components and mechanisms involved in stimulated exocytosis, including those initiated by crosslinking of IgE receptors on mast cells. To adapt this versatile assay for high throughput screening, we developed a direct, in situ method in which beta-hexosaminidase detection is carried out in a single step, convenient for multi-sample processing and thus for biosensor applications. This direct assay is efficient for measuring exocytosis in antigen-stimulated RBL mast cells, detecting antigen concentrations as low as 1 pM. We also demonstrate its utility in detecting inhibition of degranulation by a known pharmacologic inhibitor that blocks Syk tyrosine kinase activity critical for cell activation.

Animals↗
PubMed2004

Visualization of plasma membrane compartmentalization with patterned lipid bilayers.

Micrometer-size patterned lipid bilayers containing liganded lipids are used to control the location and size of receptor clusters and enable direct visualization of structural reorganization of cellular components. Subsequent to concentration of Fcepsilon receptor I, the mast cell receptor for IgE, and colocalized tyrosine phosphorylation activity, Lyn kinase and other proteins anchored to the inner leaflet of the plasma membrane redistribute selectively with the receptor clusters in a process that depends on actin polymerization. Surprisingly, outer leaflet components characteristically associated with lipid rafts do not detectably coredistribute with these inner leaflet components. Cell activation using patterned surfaces provides unique insights into cell membrane structural organization, revealing dynamic, large-scale uncoupling of inner and outer leaflet components of lipid rafts.

Actins↗
PubMed2003

Highly effective poly(ethylene glycol) architectures for specific inhibition of immune receptor activation.

Architectural features of synthetic ligands were systematically varied to optimize inhibition of mast cell degranulation initiated by multivalent crossing of IgE-receptor complexes. A series of ligands were generated by end-capping poly(ethylene glycol) (PEG) polymers and amine-based dendrimers with the hapten 2,4-dinitrophenyl (DNP). These were used to explore the influence of polymeric backbone length, valency, and hapten presentation on binding to anti-DNP IgE and inhibition of stimulated activation of RBL cells. Monovalent MPEG(5000)-DNP (IC(50) = 50 nM), bivalent DNP-PEG(3350)-DNP (IC(50) = 8 nM), bismonovalent MPEG(5000)-DNP(2) (IC(50) = 20 nM), bisbivalent DNP(2)-PEG(3350)-DNP(2) (IC(50) = 3nM) and DNP(4)-dendrimer ligands (IC(50) = 50 nM) all effectively inhibit cellular activation caused by multivalent antigen, DNP-bovine serum albumin. For different DNP ligands, we provide evidence for more effective inhibition due to (i) preferential formation of intra-IgE cross-links by bivalent ligands of sufficient length, (ii) self-association of monovalent ligands with longer tails, and (iii) higher probability of binding for bisvalent ligands. We also show that larger DNP(16)-dendrimers of higher valency trigger degranulation by cross-linking IgE-receptor complexes, whereas smaller DNP-dendrimers are inhibitory. Thus, features of synthetic ligands can be manipulated to control receptor occupation, aggregation, and inhibition of the cellular response.

Amines↗
PubMed2003

Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI.

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid-ordered properties appear to be important for their function. We investigated the possibility of using amphiphiles to disrupt lipid rafts and thereby inhibit IgE-FcepsilonRI signaling. We find that short-chain ceramides - C2-ceramide and C6-ceramide - decrease plasma membrane lipid order and reduce the extent of fluorescence resonance energy transfer between lipid-raft-associated molecules on intact cells; by contrast, biologically inactive C2-dihydroceramide does neither. Structural perturbations by these ceramides parallel their inhibitory effects on antigen-stimulated Ca2+ mobilization in RBL mast cells in the presence and absence of extracellular Ca2+. Similar inhibition of Ca2+ mobilization is caused by n-butanol, which prevents phosphatidic acid production by phospholipase D, but not by t-butanol, which does not prevent phosphatidic acid production. These results and previously reported effects of short-chain ceramides on phospholipase D activity prompted us to compare the effects of C2-ceramide, C2-dihydroceramide and C16-ceramide on phospholipase D1 and phospholipase D2 activities in vitro. We find that the effects of these ceramides on phospholipase D1 activity strongly correlate with their effects on antigen-stimulated Ca2+ mobilization and with their disruption of lipid order. Our results indicate that phospholipase D activity is upstream of antigen-stimulated Ca2+ mobilization in these cells, and they demonstrate that ceramides can serve as useful probes for investigating roles of plasma membrane structure and phospholipase D activity in cellular signaling.

Animals↗
PubMed2003

A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation.

The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcepsilonRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcepsilonRI promotes its proximity to active Lyn in a lipid raft environment.

Animals↗
PubMed2003

Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study.

Four chain spin labels and a spin-labeled cholestane were used to study the dynamic structure of plasma membrane vesicles (PMV) prepared from RBL-2H3 mast cells at temperatures ranging from 22 degrees C to 45 degrees C. Analysis shows that the spectra from most labels consist of two components. The abundant spectral components exhibit substantial ordering that is intermediate between that of a liquid-ordered (Lo) phase, and that of a liquid-crystalline (Lc) phase as represented by model membranes. Also, rotational diffusion rates of the spin labels are comparable to those in the Lo phase. In contrast, the ordering for the less abundant components is much lower. These results indicate that a Lo-like region or phase (the abundant component) and an Lc-like region or phase (the less abundant component) coexist in the PMV. In contrast, membranes reconstituted from extracted lipids exhibit the more ordered phase only. This suggests that membrane-associated proteins are important for the coexistence of Lo-like and Lc-like regions in the plasma membrane. In addition, binding of the myristoylated protein, ARF6 to PMV, leads to a new spectral component for a headgroup lipid spin label that indicates the formation of plasma membrane defects by this low molecular weight GTPase.

ADP-Ribosylation Factor 6↗
PubMed2003

Antigen-stimulated trafficking from the recycling compartment to the plasma membrane in RBL mast cells.

Binding of fluorescein isothiocyanate (FITC)-conjugated cholera toxin B subunit to ganglioside GM1 on RBL-2H3 cells at 37 degrees C results in labeling of the plasma membrane as well as a pool of perinuclear intracellular membranes identified as the endosomal recycling compartment. Antigen-mediated activation of IgE receptor signaling causes rapid, sustained outward trafficking of these labeled endosomes, that is monitored as an increase in FITC fluorescence due to relief of quenching in the acidic endosomes upon delivery to the plasma membrane. Stimulation of this process depends on the integrity of cholesterol-dependent lipid rafts and occurs in response to Ca2+-mobilizing thapsigargin as well as antigen. Inhibitors of some early signaling enzymes stimulated by FcepsilonRI, including Syk tyrosine kinase and phosphoinositide 3-kinase, have little or no effect on this trafficking response. Other signaling pathways, including activation of phospholipase C and Ca2+ influx, do not appear to be necessary for the initiation of the outward trafficking response, but they contribute to maintaining the sustained phase of this process. Consistent with this, antigen-stimulated ruffles are labeled with FITC-cholera toxin B in a Ca2+-dependent manner. Thus, this trafficking response provides a mechanism by which an internal membrane pool can contribute to plasma membrane remodeling during stimulated membrane ruffling, cell motility, and phagocytosis.

Animals↗
PubMed2002

Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI.

Degranulation of mast cells and basophils during the allergic response is initiated by Ag-induced cross-linking of cell surface IgE-Fc epsilon RI receptor complexes. To investigate how separation distances between cross-linked receptors affect the competency of signal transduction, we synthesized and characterized bivalent dinitrophenyl (DNP)-modified dsDNA oligomers with rigid spacing lengths of approximately 40-100 A. All of these bivalent ligands effectively bind and cross-link anti-DNP IgE with similar affinities in the nanomolar range. The 13-mer (dsDNA length of 44 A), 15-mer (51 A), and flexible 30-mer ligands stimulate similar amounts of cellular degranulation, about one-third of that with multivalent Ag, whereas the 20-mer (68 A) ligand is less effective and the rigid 30-mer (102 A) ligand is ineffective. Surprisingly, all stimulate tyrosine phosphorylation of Fc epsilon RI beta, Syk, and linker for activation of T cells to similar extents as multivalent Ag at optimal ligand concentrations. The magnitudes of Ca(2+) responses stimulated by these bivalent DNP-dsDNA ligands are small, implicating activation of Ca(2+) mobilization by stimulated tyrosine phosphorylation as a limiting process. The results indicate that structural constraints on cross-linked IgE-Fc epsilon RI complexes imposed by these rigid DNP-dsDNA ligands prevent robust activation of signaling immediately downstream of early tyrosine phosphorylation events. To account for these results, we propose that activation of a key downstream target is limited by the spacing between cross-linked, phosphorylated receptors and their associated components.

2,4-Dinitrophenol↗
PubMedDate not supplied

Antigen-decorated shell cross-linked nanoparticles: synthesis, characterization, and antibody interactions.

Antigen-decorated shell cross-linked knedel-like nanoparticles (SCKs) were synthesized and studied as multivalent nanoscale surfaces from which antibody-binding units were presented in a manner that was designed to approach virus particle surfaces. The SCK nanostructures were fabricated with control over the number of antigenic groups, from mixed micellization of amphiphilic diblock copolymer building blocks that contained either an antigen (2,4-dinitrophenyl) or an ethylpropionate group at the hydrophilic alpha-chain terminus. Amphiphilic diblock copolymers were synthesized by atom transfer radical polymerization of tert-butyl acrylate and methyl acrylate sequentially from either a 2,4-dinitrophenyl-functionalized initiator or ethyl 2-bromopropionate, followed by selective removal of the tert-butyl groups to afford 2,4-dinitrophenyl-poly(acrylic acid)60-b-poly(methyl acrylate)60 (DNP-PAA(60)-b-PMA60) and poly(acrylic acid)70-b-poly(methyl acrylate) (PAA70-b-PMA70). Micelles were assembled via addition of water to THF solutions of the polymers in 0:1, 1:1, and 1:0 molar ratios of DNP-PAA60-b-PMA60 to PAA70-b-PMA70, followed by dialysis against water. The acrylic acid groups of the micelle coronas were partially cross-linked (nominally 50%) with 2,2'-(ethylenedioxy)bis(ethylamine), in the presence of 1-(3'-dimethylaminopropyl)-3-ethylcarbodiimide methiodide. Following extensive dialysis against water, the 0%, 50%, and 100% dinitrophenylated shell cross-linked nanoparticles (DNP-SCKs) were characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared and UV-vis spectroscopies, and analytical ultracentrifugation (AU). The surface accessibility and bioavailability of the DNP units upon the DNP-SCKs were investigated by performing quenching titrations of fluorescein-labeled IgE antibody in solution and degranulation of IgE sensitized RBL-2H3 cells. The DNP antigens proved to be surface-available and able to form multivalent bonds with IgE antibodies, causing degranulation.

Animals↗