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B Goldstein

Publications and source records attributed to B Goldstein.

At least 19 recordsLinked to original sources
PubMed1992

Recombinant interleukin-2 analogs. Dynamic probes for receptor structure.

Interleukin-2 (IL-2) and its receptor complex have become one of the most studied members of a growing family of protein hormones characterized by structural similarities in both ligands and their receptors. Structure-function studies of IL-2 have been complicated by the multimeric nature of its receptor. Two receptor subunits (55- and 75-kDa type I cell surface proteins) can participate to form the high affinity binding site. Although the IL-2 is apparently unique in some respects, similar subunit cooperativity has now been shown to be a common feature for other members of this receptor family. The availability of cell lines expressing the individual IL-2 receptor subunits has allowed detailed analysis of subunit binding characteristics. Results regarding the relationship of molecular recognition at each subunit to the mechanism of ligand binding at the high affinity site, however, have led to different interpretations. In this study we have employed previously prepared C-terminal IL-2 mutant proteins to examine receptor binding at all three classes using a variety of equilibrium and kinetic techniques. These results indicate that the high affinity IL-2 receptor complex includes the p55/p75 heterodimer prior to IL-2 binding and that both receptor subunits participate simultaneously in ligand capture.

Binding, Competitive↗
PubMed1992

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.

Animals↗
PubMed1992

Induction of gut in Caenorhabditis elegans embryos.

Two types of developmental events can cause an embryonic cell to adopt a fate different from that of its neighbours: during a cell division particular contents may be segregated to only one daughter cell and cells may experience different external cues, commonly in the form of inductive cell interactions. Work on development in the nematode Caenorhabditis elegans suggests that most cell fates are specified without a need for cell interactions. In particular, the gut cell lineage of C. elegans has been used as a primary example of specification by differential segregation of determinants. Here I re-examine the role of induction in gut specification by isolating early blastomeres. In C. elegans, the gut derives from all the progeny of a single blastomere (E) of the eight-cell stage. When a gut precursor cell (EMS) is isolated during the first half of the four-cell stage, gut does not differentiate. Gut differentiation is rescued by recombining EMS with its posterior neighbour (P2), but not by recombining EMS with one or both of the other two cells of the four-cell stage. These results demonstrate that P2 induces EMS to form gut in C. elegans.

Animals↗
PubMed1992

Interpretation of Scatchard plots for aggregating receptor systems.

Aggregation of cell surface receptors, with each other or with other membrane proteins, occurs in a variety of experimental systems. The list of systems where receptor aggregation appears to be important in understanding ligand binding and cellular responses is growing rapidly. In this paper we explore the interpretation of equilibrium binding data for aggregating receptor systems. The Scatchard plot is a widely used tool for analyzing equilibrium binding data. The shape of the Scatchard plot is often interpreted in terms of multiple noninteracting receptor populations. Such an analysis does not provide a framework for investigating the role of receptor aggregation and will be misleading if there is a relation between receptor aggregation and ligand binding. We present a general model for the equilibrium binding of a ligand with any number of aggregating receptor populations and derive theoretical expressions for observable Scatchard plot features. These can be used to test particular models and estimate model parameters. We develop particular models and apply the general results in the cases of six aggregating receptor systems where ligand binding and receptor aggregation are related: cross-linking of monovalent cell surface proteins by monoclonal antibodies, cross-linking of cell surface antibodies by bivalent ligand, antibody-induced co-cross-linking of cell surface antibodies and Fc gamma receptors, ligand-enhanced aggregation of identical epidermal growth factor receptors, aggregation of heterologous receptors for interleukin 2 to form a high-affinity receptor, and association of receptors, including those for interleukins 5 and 6, with nonbinding accessory proteins that influence receptor affinity or effector function.

Animals↗
PubMed1992

Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are also important in shaping the binding curves. Because we have controlled experimentally for many sources of receptor heterogeneity, we have limited the potential explanations for residual Scatchard plot curvature.

Animals↗
PubMed1992

Electrophysiologic findings in post-traumatic syringomyelia: implications for clinical management.

Traumatic spinal cord injured (SCI) patients may develop pain, new weakness and/or sensory loss due to an enlarging fluid-filled cyst in the spinal cord. The clinical history and physical exam are nonspecific and insensitive, particularly for diagnosing and monitoring recurrent or progressive post-traumatic syringomyelia (PTS). We compare the sensitivity and specificity of three electrodiagnostic tests, median and ulnar F waves, electromagnetic motor evoked potentials (MEP), and needle electromyography, with respect to syrinx by imaging (MRI or CT scan) and neurologic progression on serial clinical exams. Central motor conduction time (CMCT) calculated from scalp and spine MEP was the most sensitive diagnostic test. F waves were less sensitive and less specific than the CMCT, and did not provide evidence of syrinxes in the mid or upper cervical cord. Positive sharp wave and fibrillation potentials were the least sensitive and least specific. The CMCT is a useful adjunct to imaging studies for diagnosing and monitoring PTS.

Adult↗
PubMed1992

Evidence for p55-p75 heterodimers in the absence of IL-2 from Scatchard plot analysis.

The high affinity receptor for IL-2 is composed of at least two chains, a p55 chain that binds IL-2 with low affinity and a p75 chain that binds with intermediate affinity. Two molecular mechanisms have been proposed for the formation of the high affinity receptor-ligand complex: The affinity conversion model proposes that the high affinity receptor is formed via stepwise binding in which IL-2 first binds to the p55 chain and the resulting complex then associates with the p75 chain to form a high affinity ternary complex. In the performed heterodimer model the p55 and p75 chains form a non-covalently linked high affinity heterodimer in the absence of IL-2. We show that these two models can be distinguished on the basis of equilibrium binding experiments using cell lines expressing different numbers of p55 chains. To make this distinction we develop a general model for the interaction of IL-2 with its various receptors. We than analyze the case in which heterodimers exist in the absence of IL-2 and the case in which no preformed heterodimers exist. For both cases we predict the shape of equilibrium Scatchard plots. We then show that published IL-2 binding studies are consistent with a model in which a large concentration of preformed heterodimers is present on the cell surface and inconsistent with a model in which preformed heterodimers are absent from the cell surface. The models that we develop should have general applicability to the entire class of receptor systems in which low and intermediate affinity chains interact to constitute a high affinity receptor.

Cell Membrane↗
PubMed1991

Dissociation kinetics of bivalent ligand-immunoglobulin E aggregates in solution.

We study the dissociation of preformed bivalent ligand-bivalent receptor aggregates in solution, where the ligand is a symmetric bivalent hapten with two identical 2,4-dinitrophenyl (DNP) groups and the receptor is a fluorescein-labeled monoclonal anti-DNP IgE. We promote dissociation in two ways: by the addition of high concentrations of a monovalent hapten that competes for IgE binding sites with the bivalent hapten and by the addition of high concentrations of unlabeled IgE that binds almost all ligand binding sites that dissociate from labeled IgE. We investigate both theoretically and experimentally the two types of dissociation and find them to be quite different. Theory predicts that their kinetics will depend differently on the fundamental rate constants that characterize binding and aggregation. Using monovalent ligand to promote dissociation, we find that the fraction of labeled IgE sites bound to bivalent ligand decays with a slow and fast component. The fast decay corresponds to the dissociation of a singly bound DNP hapten. The interpretation of the slow decay depends on the detailed way in which ligand-receptor aggregates break up. We show that one possible explanation of these data is that small stable rings form before the addition of monovalent ligand. Other possible explanations are also presented.

Animals↗
PubMed1991

Bivalent ligand dissociation kinetics from receptor-bound immunoglobulin E: evidence for a time-dependent increase in ligand rebinding at the cell surface.

The bivalent ligand N,N'-bis[[epsilon-[(2,4- dinitrophenyl)amino]caproyl]-L-tyrosyl]cystine [(DCT)2-Cys] binds and cross-links anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-receptor complexes on the cell surface of rat basophilic leukemia cells. The rate of dissociation of this bound ligand was monitored by using a fluorescence method under two different conditions. In one case the monovalent ligand DCT was added in large excess to prevent the dissociating ligand from rebinding to unoccupied antibody combining sites. Under these conditions, dissociation of the bivalent ligand from IgE-sensitized cells proceeds to completion with kinetics that are well described by two rate constants that are independent of the time of preincubation of the bivalent ligand with the cells. In the second case, dissociation of (DCT)2-Cys from cell-bound anti-DNP IgE was monitored in the presence of a large excess of anti-DNP IgE in solution that acts as a sink to absorb the dissociated ligand. Under these conditions, the bivalent ligand becomes more resistant to dissociation as the preincubation time of the bivalent ligand with the cells is increased. An increasing fraction of the bound ligand does not dissociate on a measurable time scale in the presence of this sink. The results indicate that cell-associated IgE-receptor complexes undergo a time-dependent change that facilitates the reformation of the cross-linked state when one end of the ligand dissociates to break up the existing cross-link. The possible physical basis and functional implications of these results are discussed.

Animals↗
PubMed1991

Effects of growth medium and cyclic AMP analogues on the cAMP-induced differentiation of F9 teratocarcinoma cells.

F9 teratocarcinoma cells differentiate into parietal endodermlike cells when treated with retinoic acid (RA) and cyclic AMP (cAMP). We have previously found that F9 cells can be induced to differentiate by treatment with cAMP in the absence of RA. In the course of determining why other investigators had failed to observe cAMP-induced differentiation, we found that the growth medium played an important role in determining the response of F9 cells to differentiating agents. When F9 cells were grown in minimal essential medium (MEM) and treated with either 8-bromo-cAMP (8BrcA) + 1-methyl, 3-isobutylxanthine (MIX), or dibutyryl cAMP (DBcA) + theophylline (T), they differentiated to parietal endodermlike cells without any requirement for exogenous RA. However, when F9 cells were grown in Dulbecco's modified Eagle's medium (DME), DBcA/T failed to induce differentiation alone and required exogenous RA to induce formation of parietal endoderm-like cells. 8BrcA/MIX alone was still able to induce some differentiation, although the extent was not as great as those cells grown in MEM. These results could not be explained by the different growth-promoting properties of the two culture media because there was no difference in the doubling time of F9 cells grown in either medium. Likewise, RA and cAMP both inhibited growth to the same extent in either medium. Inasmuch as almost all published reports on F9 cell differentiation have used DME as a growth medium, and RA with or without DBcA/T as the differentiating agents, these studies would not have had the appropriate conditions to detect the cAMP-induced differentiation of F9 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Increased intraabdominal pressure and anuria in the newborn.

Elevated intraabdominal pressure as a cause of anuria in the critically ill newborn with ascites should be considered after hypovolemia and other causes of intrinsic renal disease or obstruction have been excluded. In the rare patient with ascites who develops anuria, paracentesis should be considered if urine output cannot be established.

Anuria↗
PubMed1991

Double lumen umbilical venous catheters in critically ill neonates: a randomized prospective study.

OBJECTIVES: To compare relative efficacies and complications associated with the use of double lumen vs. single lumen umbilical venous catheters in critically ill neonates. DESIGN: Prospective randomized control trial. SETTING: Neonatal ICU. PATIENTS: Forty-three critically ill neonates. INTERVENTIONS: Group 1 patients (n = 20) received single lumen umbilical venous catheters and group 2 patients (n = 23) received double lumen catheters. A record of the following information was kept: demographic data including diagnosis and indication for umbilical venous catheter insertion, catheter tip location, length of catheterization (days), number of additional iv catheters and complications (sepsis, hepatic necrosis, thrombosis, or mechanical complications). MEASUREMENTS AND MAIN RESULTS: Double lumen umbilical venous catheters were well tolerated and were associated with no significant increased risk of mechanical complications when compared with single lumen umbilical venous catheters. The number of additional iv catheters required (0.8 +/- 0.1 [SD]) was significantly (p less than .05) less in the double lumen umbilical venous catheter group as compared with additional iv catheters required (2.3 +/- 0.8) in the single lumen umbilical venous catheter group. CONCLUSION: Double lumen umbilical venous catheters are well tolerated for short-term use, decrease the need for additional venous catheters in critically ill neonates, and may not significantly increase the risk of mechanical complications when compared with single lumen umbilical venous catheters.

Catheterization, Central Venous↗
PubMed1990

Cross-linking of Fc gamma receptors and surface antibodies. Theory and application.

B lymphocyte responses to the cross-linking of surface Ig (sIg) are known to be inhibited, when IgG is the cross-linking agent, by the concurrent binding of the Fc portion of the IgG to Fc gamma R. We present a mathematical framework for designing and analyzing experiments aimed at uncovering the inhibition mechanism(s). From our model, we calculate concentrations of receptors and ligands in the different cell surface states, at equilibrium or as a function of time. IgG can cross-link surface receptors in three ways, i.e., by bridging two sIg molecules without Fc binding, by bridging two sIg while binding as well to an Fc gamma R, and by binding to an Fc gamma R and only one sIg. We show how the concentrations or fractions of these distinct cross-linked states depend on experimentally manipulable variables, including the concentrations of intact IgG, bivalent and monovalent IgG fragments, and agents that block Fc binding. Then, using simple signal/response relationships, reflecting active and passive mechanisms of Fc-mediated inhibition, we simulate the results of a variety of experiments. In cases where published experimental results are available, we find that the qualitative predictions of our general model are consistent with the data and that comparisons of simulations with available data provide some quantitative information about the parameters governing the cell surface signaling events. In particular, comparison of model predictions with published experiments on the kinetics of IgG-induced inositol trisphosphate production indicate that sIg cross-links form more rapidly than sIg-Fc gamma R "co-cross-links." Further, IgG-sIg bonds stabilize Fc attachments, i.e., the dissociation of IgG from Fc gamma R is slowed significantly when the IgG is also cross-linked to sIg. Predictions of the model suggest other experiments and ways of presenting the data that will help to identify relationships between the molecular signaling events occurring on the cell surface and the various cellular responses.

Animals↗
PubMed1990

Cyclic adenosine monophosphate-mediated induction of F9 teratocarcinoma differentiation in the absence of retinoic acid.

F9 teratocarcinoma stem cells differentiate into parietal endoderm-like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB-cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm-like cells in the absence of exogenous RA when treated with cholera toxin and 1-methyl,3-isobutyl xanthine (CT/MIX) or 8-bromo-cAMP/MIX (8B2-cAMP/MIX). Cells treated with CT/MIX or 8B2-cAMP/MIX were morphologically similar to parietal endoderm-like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage-specific embryonic antigen (SSEA-1) and an mRNA species of 6.8 kb (pST6-135) were markedly reduced in CT/MIX-treated cells. To prove that cAMP alone could induce differentiation Lipidex-1000, a hydrophobic gel, was used to remove 80-90% of the endogenous serum retinoids. F9 cells grown in this retinoid-depleted serum and treated with 8B2-cAMP/MIX differentiated to parietal endoderm-like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm-like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.

1-Methyl-3-isobutylxanthine↗