Biomedical subjects

B Baird

Publications and source records attributed to B Baird.

At least 73 records · Page 4Linked to original sources

PubMed1990

Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig.

We have investigated the interaction of mouse (m) IgE with its Fc epsilon RI on rat basophilic leukemia cells using a set of chimeric Ig that were constructed by exchanging homologous H chain C domains between human (hu) IgG1 and mIgE. Binding affinities were examined with equilibrium and kinetic measurements, and we found that epsilon/C gamma 3 (mIgE with C epsilon 4 replaced by C gamma 3) was indistinguishable from mIgE. The huIgG1 and the other chimeric Ig, which did not contain both C epsilon 2 and C epsilon 3, did not bind detectably to rat basophilic leukemia cells (Ka less than 10(6) M-1). The ability of these chimeric Ig to stimulate a cellular response (degranulation) in the presence of multivalent Ag was also tested. The epsilon/C gamma 3 was indistinguishable from mIgE in eliciting a high level of degranulation, whereas the other chimeric Ig stimulated no response even when they were preaggregated to enhance their binding avidity. These results demonstrate that C epsilon 4 may be replaced by C gamma 3 without affecting the binding and cell activating properties of mIgE. The lack of binding by the other chimeric Ig indicates that both C epsilon 2 and C epsilon 3 are necessary for the binding interaction.

Animals↗

PubMed1990

Microfilaments regulate the rate of exocytosis in rat basophilic leukemia cells.

Disruption of microfilaments in rat basophilic leukemia (RBL) cells by exposure to cytochalasin B is observed to potentiate the rate of antigen-stimulated secretion from these cells. Under these conditions, cytochalasin B is without effect on the antigen-stimulated production of inositol phosphates or 45Ca2(+)-influx. In streptolysin-O-permeabilized RBL cells, cytochalasin B is observed to potentiate the rate of secretion in response both to guanosine 5'-(2-thio)-O-triphosphate (GTP gamma S) and to Ca2+ (buffered between 0.1 and 10 microM). However, under these conditions, cytochalasin B does not affect to antigen-stimulated production of inositol phosphates. Consistent with these data, microfilaments are proposed to regulate a terminal step in exocytosis, in a physiologically relevant manner.

Actin Cytoskeleton↗

PubMed1990

A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E.

Time-resolved fluorescence anisotropy measurements have been used to examine the segmental flexibility of anti-dansyl immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells. Although IgE in this complex exhibits only a restricted angular range of segmental motion, much of this restricted motion occurs on a relatively rapid time scale. A fast component of motion with a rotational correlation time of 15-35 ns may correspond to the twisting of Fab segments about their major axis. Intermolecular cross-linking by a short bivalent ligand, N,N'-didansylcadaverine, results in complete loss of this segmental motion. Solubilization of monomeric IgE-receptor complexes using a zwitterionic detergent results in a time-dependent anisotropy decay that exhibits both a fast component and a slower component that is intermediate between the decay for soluble and membrane-bound forms of IgE at long times after excitation. These results are discussed in terms of a model in which binding of IgE to its membrane-bound receptor restricts not only its global rotation but also its slower modes of segmental flexibility as well, while allowing its Fab segments to undergo rapid reorientation within a limited angular range.

Animals↗

PubMed1990

A guanine nucleotide-binding protein participates in IgE receptor-mediated activation of endogenous and reconstituted phospholipase A2 in a permeabilized cell system.

Activation of phospholipase A2 (PLA2) by the aggregation of receptors for immunoglobulin E (IgE) can be studied in streptolysin O-permeabilized rat basophilic leukemia cells. Under these conditions, 40 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) stimulates PLA2 activity 5-6-fold when free Ca2+ concentrations are buffered at 10(-7)-10(-5) M. Antigen-mediated cross-linking of receptors for IgE synergizes with low concentrations of GTP gamma S (0.1 microM) to cause similar stimulation. When the endogenous PLA2 activity is inactivated by chemical modification, we find that exogenously supplied PLA2 from porcine pancreas and Naja naja venom is also activated by the aggregation of cell-surface IgE receptors in these permeabilized cells. As with endogenous PLA2, GTP gamma S synergizes with IgE receptor-aggregation to activate exogenous PLA2 approximately 10-fold at 10(-7)-10(-6) M free Ca2+. These data indicate that receptor-mediated activation of a guanine nucleotide-binding protein can shift the Ca2+ dependence of PLA2 activity resulting in greatly enhanced activity at physiological concentrations of intracellular free Ca2+. The partial reconstitution of various PLA2 forms into such a broken-cell system offers a new approach for studying the mechanisms of G-protein-mediated activation of PLA2.

Animals↗

PubMed1990

Characterization of model antigens composed of biotinylated haptens bound to avidin.

Model antigens of defined structure and limited valency have been prepared by binding 2,4-dinitrophenyl (DNP)-biotin haptens of different lengths to the biotin binding sites of avidin. These complexes have been characterized structurally by spectroscopic methods and functionally by testing their ability to crosslink cell-bound anti-DNP immunoglobulin E-receptor complexes to stimulate degranulation of rat basophilic leukemia cells. One of these haptens, 1-DNP-amino-12-biotinamido dodecane, is shown to have only two tight binding sites per avidin molecule, and the resulting bivalent (DNP-biotin)-avidin antigen is found to stimulate substantial degranulation. Another DNP-biotin hapten that is approximately 10 A longer has four tight binding sites per avidin and, when bound to avidin, has greater activity similar to a highly DNP-conjugated multivalent antigen. These and other (DNP-biotin)-avidin complexes described here represent a new group of chemically well-defined antigens that are useful for studying the binding to and functional crosslinking of cell-bound immunoglobulins in immunological responses.

Animals↗

PubMed1989

Bivalent binding of an anti-CD3 antibody to Jurkat cells induces association of the T cell receptor complex with the cytoskeleton.

Ligand binding and cross-linking of TCR/CD3 complex leads to T cell stimulation in immunologic responses. As a prelude to investigating the dynamic interactions of these receptors, we have characterized binding of the mAb OKT3 specific for CD3 on Jurkat cells. The association of both OKT3 and its Fab' fragment is rapid at 4 degrees C, and dissociation of Fab' is also rapid, but dissociation of OKT3 is slow, indicating bivalent binding in this case. Dissociation of OKT3 is substantially accelerated at 37 degrees C if internalization is prevented. From the concentration dependence, the binding of OKT3 at 4 degrees C appears to be very tight whereas binding of the Fab' fragment is weaker and biphasic. Scatchard analysis of the Fab' equilibrium binding data indicates two binding sites with KD values of 5.1 x 10(-9) M and 2.7 x 10(-8) M. The very tight binding of the bivalent antibody may be caused by inter- or intramolecular cross-linking between these sites. If Jurkat cells are warmed to 37 degrees C, there is an energy-dependent increase by about one-third of sites bound by OKT3 or its Fab' fragment over that seen at 4 degrees C. This increase may be related to a receptor recycling process because internalization of a similar number of the bound ligands occurs at similar rates. Other experiments have revealed that OKT3, but not its Fab' fragment, causes the receptor complex to become associated with the detergent-insoluble cytoskeleton, and there are also insoluble intracellular OKT3-binding sites. These cross-linking-induced receptor-cytoskeletal interactions are sensitive to moderate changes in salt concentration that should allow their molecular basis to be investigated.

Antibodies, Monoclonal↗

PubMed1989

Antigen-triggered membrane potential changes in IgE-sensitized rat basophilic leukemia cells: evidence for a repolarizing response that is important in the stimulation of cellular degranulation.

We have studied Ag-induced membrane potential changes of rat basophilic leukemia cells by using the potential-sensitive dye, bis-(1,3-diethylthiobarbiturate)trimethineoxonol. A rapid membrane depolarization is triggered by a multivalent Ag, and it has a bell-shaped dose dependence that parallels the degranulation response but not the extent of cross-linking of the IgE-receptor complexes. As the temperature is reduced from 37 degrees C, this depolarization response slows and decreases in magnitude until complete inhibition is observed at 15 degrees C, similar to the temperature dependence previously observed for the Ag-stimulated rise in cytoplasmic Ca2+ and for degranulation. The results imply that a highly temperature-dependent step subsequent to Ag binding and cross-linking is necessary for the depolarization response. A partial return to the resting potential is seen to follow the depolarization response to Ag. This repolarization process is inhibited by quinidine.HCl and Ba2+ in parallel with an inhibition of the degranulation response. Repolarization is not affected by 4-aminopyridine or by the absence of K+ in the external buffer. These data suggest that the repolarization is caused by a previously uncharacterized K+ channel.

Animals↗

PubMed1989

Competition between solution and cell surface receptors for ligand. Dissociation of hapten bound to surface antibody in the presence of solution antibody.

We present a joint theoretical and experimental study on the effects of competition for ligand between receptors in solution and receptors on cell surfaces. We focus on the following experiment. After ligand and cell surface receptors equilibrate, solution receptors are introduced, and the dissociation of surface bound ligand is monitored. We derive theoretical expressions for the dissociation rate and compare with experiment. In a standard dissociation experiment (no solution receptors present) dissociation may be slowed by rebinding, i.e., at high receptor densities a ligand that dissociates from one receptor may rebind to other receptors before separating from the cell. Our theory predicts that rebinding will be prevented when S much greater than N2Kon/(16 pi 2D a4), where S is the free receptor site concentration in solution, N the number of free surface receptor sites per cell, Kon the forward rate constant for ligand-receptor binding in solution, D the diffusion coefficient of the ligand, and a the cell radius. The predicted concentration of solution receptors needed to prevent rebinding is proportional to the square of the cell surface receptor density. The experimental system used in these studies consists of a monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), that reversibly binds to a monoclonal anti-DNP immunoglobulin E (IgE). This IgE is both a solution receptor and, when anchored to its high affinity Fc epsilon receptor on rat basophilic leukemia (RBL) cells, a surface receptor. For RBL cells with 6 x 10(5) binding sites per cell, our theory predicts that to prevent DCT rebinding to cell surface IgE during dissociation requires S much greater than 2,400 nM. We show that for S = 200-1,700 nM, the dissociation rate of DCT from surface IgE is substantially slower than from solution IgE where no rebinding occurs. Other predictions are also tested and shown to be consistent with experiment.

Animals↗

PubMed1989

Interaction of IgE with its high-affinity receptor. Structural basis and requirements for effective cross-linking.

Structural interactions between IgE and its high-affinity receptor have been investigated with the methods of fluorescence resonance energy transfer and genetic engineering. The results indicate that IgE has a bent conformation when bound to receptor on the cell surface and that the site of interaction is contained in the C epsilon 2 and C epsilon 3 domains; the C-terminal domain, C epsilon 4, is not required for binding. Cross-linking of IgE-receptor complexes is required for signal transduction across the plasma membrane. Binding studies with defined bivalent ligands indicate that structural and/or kinetic features determine the functional effectiveness of the cross-linked states.

Animals↗

PubMed1989

Proteolytic digestion of the beta and gamma subunits of the receptor for immunoglobulin E at the cytoplasmic face of the plasma membrane.

Cytoplasmically exposed portions of the high affinity receptor for immunoglobulin E were investigated with controlled proteolytic digestion of plasma membrane vesicles from rat basophilic leukemia cells. Hypotonic shock treatment results in vesicle inversion, thereby exposing the cytoplasmic portions of the approximately 32 kDa beta and approximately 8 kDa gamma subunits to surface labeling by lactoperoxidase-catalyzed 125I-iodination. These 125I-labeled protein segments disappeared after treating inverted vesicles with trypsin, and labeled components also disappeared when chloramine T mediated 125I-iodination was used to label receptors after inverted vesicles had been trypsin digested and solubilized. Biosynthetic labeling of receptors with 35S-methionine showed that a 17-19 kDa labeled fragment, designated beta', remains associated with alpha after trypsin digestion of inverted vesicles. This beta' fragment was confirmed to be the intramembranous portion of the beta subunit in experiments where receptors were labeled with the hydrophobic photoactivated probe 3-(trifluoro-methyl)-3-(m-[125I] iodophenyl)diazirine prior to digestion. Our experimental results are consistent with the amino acid sequence and topography of the beta subunit predicted from the recently cloned cDNA for this subunit (Kinet, J.-P., et al., Proc. Natl. Acad. Sci. 85, 6483, 1988). Since the cytoplasmically-exposed portions of the beta and gamma subunits can also be efficiently and selectively removed from solubilized receptors by trypsin, reconstitution experiments to examine the importance of these segments in mediating the delivery of the transmembrane signal are made possible.

Animals↗

PubMed1988

Cholera toxin increases the rate of antigen-stimulated calcium influx in rat basophilic leukemia cells.

Cholera toxin pretreatment has been found to cause a 3-fold increase in the initial rate of antigen-stimulated secretion of serotonin from rat basophilic leukemia (RBL) cells. Under similar conditions, cholera toxin enhances the antigen-stimulated rise in cytoplasmic free ionized calcium levels and causes a 2-3-fold increase in the rate of antigen-stimulated influx of 45Ca. In intact RBL cells cholera toxin pretreatment potentiates the antigen-stimulated production of inositol phosphates, but in permeabilized cells, with strongly buffered free calcium levels, no effect of cholera toxin pretreatment on the antigen-stimulated activation of cellular phospholipase activities is observed. In addition, pretreatment of cells with tetradecanoylphorbol acetate inhibits antigen-stimulated production of inositol phosphates by greater than 95%, while the stimulated influx of 45Ca remains unaffected. These data indicate that the antigen-stimulated influx of calcium into RBL cells can be dissociated from the production of inositol phosphates in these cells. The observed effects of cholera toxin on exocytosis and Ca2+ influx in RBL cells are not due to the elevation of cellular cyclic AMP levels since a variety of agents capable of elevating cellular cyclic AMP levels do not mimic these effects. Together, these data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the pathway responsible for the antigen-stimulated influx of calcium into RBL cells.

Animals↗

PubMed1988

Molecular crowding on the cell surface.

Strong steric interactions among proteins on crowded living cell surfaces were revealed by measurements of the equilibrium spatial distributions of proteins in applied potential gradients. The fraction of accessible surface occupied by mobile surface proteins can be accurately represented by including steric exclusion in the statistical thermodynamic analysis of the data. The analyses revealed enhanced, concentration-dependent activity coefficients, implying unanticipated thermodynamic activity even at typical cell surface receptor concentrations.

Animals↗

PubMed1988

Cross-linking of IgE-receptor complexes by rigid bivalent antigens greater than 200 A in length triggers cellular degranulation.

We have examined the effect of cross-linking IgE-receptor complexes with variable receptor-receptor distances on the transmembrane signaling that leads to degranulation of rat basophilic leukemia cells. Linear polymers of the biotin-binding protein avidin were generated with bis biotin-1,12-diamidododecane, and a dinitrophenyl-biotin conjugate was bound at each end of the polymers to form a series of rigid bivalent haptens of well-defined length. The polymers were fractionated by size with nondenaturing PAGE, electro-eluted, and tested for their ability to stimulate degranulation of rat basophilic leukemia cells sensitized with anti-DNP IgE. We found that hexamers of avidin (of length greater than or equal to 240 A) were as effective in triggering degranulation as dimers (of length approximately 80 A), while the monomeric avidin antigen (of length approximately 40 A) elicited a poorer degranulation response from the cells. The mechanism by which aggregation of cell surface receptors can initiate signal transduction is discussed in light of these results.

Animals↗

PubMed1988

Treatment of Pneumocystis carinii pneumonia with trimetrexate in acquired immunodeficiency syndrome (AIDS).

In vitro studies have shown that trimetrexate, a lipid-soluble analogue of methotrexate, is 1500 times more potent than trimethoprim as an inhibitor of dihydrofolate reductase from Pneumocystis carinii. Furthermore, trimetrexate is readily taken up by P carinii, while performed folates such as leucovorin are not. These observations suggest that the combination of trimetrexate plus leucovorin, which can specifically protect mammalian host tissues from the toxic effects of the antifolate, may be useful in the treatment of pneumocystis pneumonia. This concept was tested in a clinical study of 49 patients with acquired immunodeficiency syndrome (AIDS) and P carinii pneumonia who were treated for 21 days with trimetrexate and leucovorin. Patients were divided into three groups: 16 patients who were unable to tolerate or had failed both pentamidine isethionate and trimethoprim-sulfamethoxazole therapy were treated with trimetrexate plus leucovorin (Group I); 16 patients who were unable to tolerate sulfonamide therapy were treated with trimetrexate with leucovorin as initial therapy (Group II); and 17 patients in whom trimetrexate with leucovorin plus sulfadiazine was used as initial therapy (Group III). Response and survival rates were 69% and 69% in Group I; 63% and 88%, respectively, in Group II; and 71% and 76%, respectively, in Group III. Toxicity was minimal. The results indicate that trimetrexate with leucovorin is safe and effective for initial therapy in AIDS patients with P carinii pneumonia and in those intolerant or unresponsive to standard therapies.

Acquired Immunodeficiency Syndrome↗

PubMed1987

Trimetrexate for the treatment of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome.

Preclinical studies have demonstrated that trimetrexate is a potent inhibitor of dihydrofolate reductase from Pneumocystis carinii. On the basis of this evidence, this lipid-soluble antifolate was used as an antipneumocystis agent in 49 patients with the acquired immunodeficiency syndrome (AIDS) and pneumocystis pneumonia. Simultaneous treatment with the reduced folate leucovorin was used as a specific antidote to protect host tissues from the toxic effects of the antifolate without affecting the antipneumocystis action of trimetrexate. Patients were assigned to three groups and treated for 21 days: in Group I, trimetrexate with leucovorin was used as salvage therapy in patients in whom standard treatments (both pentamidine isethionate and trimethoprim-sulfamethoxazole) could not be tolerated or had failed (16 patients); in Group II, trimetrexate with leucovorin was used as initial therapy in patients with a history of sulfonamide inefficacy or intolerance (16 patients); and in Group III, trimetrexate with leucovorin plus sulfadiazine was used as initial therapy (17 patients). The response and survival rates were, respectively, 69 percent and 69 percent in Group I; 63 percent and 88 percent in Group II; and 71 percent and 77 percent in Group III. Trimetrexate therapy had minimal toxicity; transient neutropenia or thrombocytopenia occurred in 12 patients and mild elevation of serum aminotransferases in 4. We conclude that the combination of trimetrexate and leucovorin is safe and effective for the initial treatment of pneumocystis pneumonia in patients with AIDS and for the treatment of patients with intolerance or lack of response to standard therapies.

Acquired Immunodeficiency Syndrome↗

PubMed1987

The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors.

For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.

Animals↗

PubMed1987

Relation of olfactory EEG to behavior: spatial analysis.

The hypothesis that odor-specific patterns of mitral cell activity during odor discrimination might be found in the corresponding spatial patterns of electroencephalogram (EEG) amplitude over a surgically accessible segment of the bulbar surface was tested in rabbits with chronically implanted electrode arrays. The spatial spectrum of the bulbar EEG was derived and compared with the spectrum predicted for the granule cell generator. Spatial filters were devised to identify, enhance, or remove the granule cell contribution to the EEG. Spatial deconvolution was applied to the filtered granule cell activity patterns to correct for distortion caused by volume conduction. The results indicated that the bulb generated odor-specific spatial patterns in rabbits trained to discriminate between two odors. The odor-specific information was not localizable to subsets of channels. This suggested that the discriminative output of the bulb involved the entire structure, even though the receptor input was delivered to limited subsets of mitral cells.

Animals↗

PubMed1987

Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation.

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.

Animals↗