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S C Dreskin

Publications and source records attributed to S C Dreskin.

At least 19 recordsLinked to original sources
PubMed2001

In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain.

MEKK2 and MEKK3 are mitogen-activated protein kinase kinase kinases (MAP3 kinases) of 70 and 71 kDa respectively that are markedly homologous (94%) in their kinase domains. Both MEKK2 and MEKK3 are able to activate the Jun kinase pathway in vivo. However, following routine immunoprecipitation in Triton X-100, MEKK2 but not MEKK3 is able to effectively phosphorylate both SEK-1 and MEK-1 and to undergo autophosphorylation. Unexpectedly, both MEKK2 and MEKK3 are functional in an in vitro kinase assay when cells are solubilized with the closely related detergent, NP-40. Given the high homology between these kinases, we set out to relate this differential sensitivity to Triton X-100 to differences in primary structure. A set of chimeric molecules were generated and the loss of activity in Triton X-100 mapped to kinase domain II/III and specifically to serine 390 of MEKK3 and valine 384 of MEKK2, residues immediately N-terminal to the active site lysine. Mutation of serine 390 of MEKK3 to a valine (as is found in MEKK2) conferred catalytic activity to MEKK3 in Triton X-100 whereas the reciprocal alteration of valine 384 of MEKK2 to a serine conferred lack of activity in Triton X-100 to MEKK2. Search of the protein database identified only three kinases, MEKK3, Pbs2p and Dd-PKI, with a serine or threonine at this site. The presence of a serine or threonine adjacent to the active site lysine in protein kinases is rare and, in MEKK3, results in detergent instability.

Amino Acid Sequence↗
PubMed2001

Isoforms of Jun kinase are differentially expressed and activated in human monocyte/macrophage (THP-1) cells.

Ten isoforms of c-jun N-terminal kinase (JNK) have been described that arise by differential mRNA splicing of three genes. In that the relative expression and function of these different JNK proteins in human monocytic cells is not known, we have examined the JNK isoforms in THP-1 monocyte/macrophage cells. Differentiation of THP-1 cells by exposure to 10(-8) M PMA for 42-48 h enhances cellular responses to LPS, including enhanced activation of total JNK activity and increased phosphorylation of p54 JNK as well as p46 JNK. Examination of JNK proteins on Western blots reveals a predominance of p46 JNK1 and p54 JNK2 proteins. Clearing of lysates by immunoprecipitation of JNK1(99% effective) removes 46% of the JNK enzymatic activity (p < 0.01), whereas clearing of JNK1 plus JNK2 (70% effective) depletes the sample of 72% of the JNK activity (p < 0.01). Further analysis, undertaken with real-time RT-PCR, revealed that 98% of the JNK messages code for three isoforms: JNK1beta1, JNK2alpha1, and JNK2alpha2. The p54 JNK that is phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is most likely JNK2alpha2 because 97% of the p54 JNK-encoding messages code for JNK2alpha2. By analogous reasoning, the p46 JNKs that are not heavily phosphorylated, but account for approximately half of the N-terminal c-jun kinase enzymatic activity, are most likely either JNK1beta1 or JNK2alpha1 because they account for 98% of the messages that can code for 46kDa JNKS:

Blotting, Western↗
PubMed1999

A diabetogenic gene prevents T cells from receiving costimulatory signals.

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.

Animals↗
PubMed1999

Production of TNF-alpha by murine bone marrow derived mast cells activated by the bacterial fimbrial protein, FimH.

Production of tumor necrosis factor alpha (TNF-alpha) by mast cells is an important aspect of host defense against gram negative bacteria. In order to define the intracellular pathways utilized by mast cells in this physiological, protective role, we have studied the production of TNF-alpha in bone marrow derived mast cells from the C3H/HeJ (LPS-insensitive) strain following exposure to bacteria expressing the fimbrial protein, FimH. Mast cells exposed to FimH produce TNF-alpha (300-1200 pg/10(6) cells) over 1-3 h compared with 1800-15,000 pg/10(6) cells produced by cells triggered via IgE/antigen. This low level of TNF-alpha production in vitro is compatible with the protective in vivo role of mast cells to produce modest amounts of TNF-alpha in contrast to the large amounts of mediators released during maximal activation. A second difference between the two signals is sensitivity to cyclosporin A (CsA). The IgE/antigen pathway is inhibited by 90-95% at 0.02 to 0.5 microM cyclosporin A whereas the FimH pathway is inhibited by only 40%. These data demonstrate that the intracellular pathway activated by FimH is different from that activated by IgE/antigen both in terms of amount of TNF-alpha produced and in sensitivity to CsA. This is the first evidence that FimH activates mast cells via a pathway that is distinct from that used by IgE/antigen.

Adhesins, Bacterial↗
PubMed1996

Arachidonate-induced eicosanoid synthesis in RBL-2H3 cells: stimulation with antigen or A23187 induces prolonged activation of 5-lipoxygenase.

We studied the ability of rat basophilic leukemia (RBL-2H3) cells stimulated with either IgE/antigen or calcium ionophore, A23187, to synthesize LTC4 and PGD2 after addition of exogenous arachidonic acid. RBL-2H3 cells preferentially synthesized PGD2 in response to stimulation with low concentrations of antigen or A23187 while higher concentrations also resulted in a marked synthesis of LTC4. The synthesis of LTC4 was dependent upon initial activation of 5-lipoxygenase by IgE/antigen or A23187, since arachidonic acid lone failed to induce LTC4 synthesis. Following the addition of IgE/antigen or A23187 alone, the synthesis of PGD2 and LTC4 was essentially complete by 10 min. To determine whether a limitation of substrate precluded further eicosanoid synthesis, exogenous arachidonic acid was added to washed cells 15-145 min following the initial stimulation with IgE/antigen or A23187, PGD2 and LTC4 synthesis was resumed following the addition of arachidonic acid to washed prestimulated cells, demonstrating that the termination of eicosanoid synthesis in RBL-2H3 cells was nor caused by the inactivation of cyclooxygenase and 5-lipoxygenase. DNP-lysine was added to cells previously stimulated with IgE/antigen to stop receptor aggregation and this greatly inhibited subsequent production of LTC4 following the addition of arachidonic acid, suggesting that ongoing stimulation of Fc epsilon XsRI was required for LTC4 synthesis in this setting. These results indicate that the magnitude of a physiologic stimulus (IgE/antigen) can profoundly affect the arachidonate metabolites produced by mast cells and that the synthesis of these metabolites quickly becomes limited by substrate availability rather than the activity of cyclooxygenase or 5-lipoxygenase.

Animals↗
PubMed1996

High-affinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I.

Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we have identified oligonucleotides that bind to human IgE with high affinities and high specificity. These ligands were isolated from three pools of oligonucleotides, each representing 10(15) molecules: two pools contained 2'-NH2 pyrimidine-modified RNA with either 40 or 60 randomized sequence positions, and the third pool contained ssDNA with 40 randomized sequence positions. Based on sequence and structure similarities, these oligonucleotide IgE ligands were grouped into three families: 2'-NH2 RNA group A ligands are represented by the 35-nucleotide truncate IGEL1.2 (Kd = 30 nM); 2'-NH2 RNA group B ligands by the 25-nucleotide truncate IGEL2.2 (Kd = 35 nM); and the ssDNA group ligands by the 37-nucleotide truncate DI 7.4 (Kd = 10nM). Secondary structure analysis suggests G quartets for the 2'-NH2 RNA ligands, whereas the ssDNA ligands appear to form stem-loop structures. Using rat basophilic leukemia cells transfected with the human high-affinity IgE receptor Fc epsilon RI, we demonstrate that ligands IGEL1.2 and D17.4 competitively inhibit the interaction of human IgE with Fc1 epsilon RI. Furthermore, this inhibition is sufficient to dose-dependently block IgE-mediated serotonin release from cells triggered with IgE-specific Ag or anti-IgE Abs. Therefore, these oligonucleotide ligands represent a novel class of IgE inhibitors that may prove useful in the fight against allergic diseases.

Antibody Affinity↗
PubMed1995

ATP-dependent activation of phospholipase C by antigen, NECA, Na3VO4, and GTP-gamma-S in permeabilized RBL cell ghosts: differential augmentation by ATP, phosphoenolpyruvate and phosphocreatine.

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with alpha-toxin from S. aureus are a simplified system for the study of Fc epsilon RI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP-gamma-S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to Fc epsilon RI and GTP-gamma-S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na3VO4) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP-gamma-S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na3VO4 is augmented by PEP and PCr in a fashion similar to that observed for Fc epsilon RI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by Fc epsilon RI or Na3VO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1995

A method to trap IgG antibodies within functional membrane vesicles.

Monoclonal antibodies have been used extensively to study the roles of specific extracellular proteins in cellular activation. Due to the large size of these molecules, their use for the study of receptor-mediated activation of intracellular processes has been limited. This report describes a method to introduce whole IgG antibody molecules into large functional membrane vesicles (ghosts) derived from rat basophilic leukemia (RBL) cells. Furthermore, an IgG1 mouse monoclonal antibody (JRK) directed against the cytoplasmic portion of the beta subunit of the high affinity receptor for IgE (Fc epsilon RI) can partially inhibit Fc epsilon RI-mediated PI hydrolysis through modest effects on both basal and receptor-mediated activation of phospholipase C.

Animals↗
PubMed1993

Phosphoenolpyruvate and creatine phosphate augment ATP and magnesium-dependent, Fc epsilon RI-mediated activation of phospholipase C in RBL cell ghosts.

We have previously reported that Fc epsilon RI-mediated activation of PLC occurs in plasma membrane vesicles derived from RBL cells. This activity is dependent on ATP and magnesium, and is greatly enhanced by the addition of either PEP or CP. We undertook these studies to determine whether these compounds augment Fc epsilon RI-mediated activation of PLC because of their ability to form ATP. The specific findings were 1) the addition of increasing amounts of ATP to the ghost vesicles cannot substitute for the combination of ATP and either PEP or CP; 2) the addition of increasing amounts of ATP or a combination of ATP and either PEP or CP results in similar levels of intravesicular ATP; 3) many metabolically related compounds have some activity to support receptor-mediated PI hydrolysis, but only PEP, CP, and 3-PG give a maximal signal; and 4) the inability of ATP alone to support optimal receptor-mediated PI hydrolysis does not appear to be due to the accumulation of ADP is only modestly inhibitory. Taken together, these data are evidence that augmentation of Fc epsilon RI-mediated PI hydrolysis by PEP and CP is not explained simply by the ability of these compounds to generate intravesicular ATP.

Adenosine Triphosphate↗
PubMed1993

Fc epsilon RI-mediated hydrolysis of phosphoinositides in permeable membrane vesicles.

We have used hypotonic lysis of cytoplasts derived from rat basophilic leukemia (RBL) cells to prepare organelle and cytoplasm-depleted membrane vesicles called 'RBL cell ghosts' (Dreskin and Metzger, 1991). Unlike other membrane preparations, the RBL ghosts hydrolyze phosphoinositides (PIs) in response to aggregation of the high affinity receptor for IgE (Fc epsilon RI) and have proven useful for studies of the molecular events involved in transduction of this signal. A significant limitation of these preparations is that they are sealed. Thus, to incorporate membrane-impermeant molecules (such as ATP) into the intravesicular space of the ghosts, they must be added as the ghosts are formed. We have now overcome this problem by permeabilizing the ghosts with alpha-toxin from S. aureus and find that, following permeabilization, ghosts activated via Fc epsilon RI, hydrolyze PIs for a longer time than do non-permeabilized ghosts. As in the intact ghosts, this response is absolutely dependent upon ATP and is enhanced by the addition of either phosphoenolpyruvate (PEP) or creatine phosphate (CP). This report demonstrates that we can now manipulate the intravesicular environment of the RBL ghosts and extends the utility of these preparations as a model system for the study of signal transduction following activation via Fc epsilon RI.

Adenosine Triphosphate↗
PubMed1993

Fc epsilon RI-mediated expression of mRNA for c-fos in rat basophilic leukemia cells does not require ongoing aggregation of the receptor.

Cross-linkage of the high-affinity receptor for IgE (Fc epsilon RI) by a polyvalent ligand, leads to activation of mast cells and basophils. We have studied Fc epsilon RI-mediated expression of RNA coding for the protooncogene, c-fos, in rat basophilic leukemia (RBL) cells and specifically have examined the requirements for ongoing receptor aggregation in the generation of this signal. RBL cells were sensitized with IgE specific for 2,4-dinitrophenyl (DNP) and incubated at 37 degrees C in the presence of DNP24BSA or BSA alone. Following activation for 0 to 30 min, the reaction was terminated. RNA was isolated and separated on denaturing gels, blotted to nylon membranes and hybridized with a 32P-labelled cDNA probe for c-fos. Messenger RNA for c-fos is detectable as early as 5-10 min following the addition of antigen and increases in a time-dependent fashion over 30 min. Unexpectedly, the addition of the hapten, 10(-4) M DNP-lysine, 5 min after the addition of antigen (which causes immediate cessation of exocytosis) does not dramatically alter the amount of message detected at 30 min. This effect is present as early as 2 min after cross-linking of the receptor and occurs at various doses of the aggregating stimulus. Thus, in contrast to the case with exocytosis and other well-described intracellular events, Fc epsilon RI-mediated increases in the level of mRNA for c-fos does not require ongoing aggregation of Fc epsilon RI.

Animals↗
PubMed1991

Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.

Adenosine Triphosphate↗
PubMed1989

A staphylococcal protein A rosetting assay for the demonstration of high affinity IgE receptors on rIL-3-dependent human basophil-like cells grown in mixed cell cultures.

Mature basophils can be differentiated from other blood born cells by their morphology, metachromatically staining granules, histamine content, and the presence on their surfaces of high-affinity IgE receptors. The identification of basophil-like cells arising in mixed human bone marrow cultures, however, is made more difficult because of their immaturity and the morphologic alterations due to in vitro culture. Identification of IgE receptors on individual cells in cell preparations which could simultaneously be examined for their histochemical properties would facilitate the study of basophil growth and differentiation. Because this was difficult using existing techniques, we developed an IgE staphylococcal protein A rosetting assay which allows the identification of cells bearing high-affinity IgE receptors and permits the same cells to be examined by a variety of staining techniques. We then examined the appearance of basophil-like cells bearing IgE receptors in cultures of human bone marrow and correlated this data with measurements of IgE receptor number and affinity. The percentage of total cells grown in the presence of human recombinant IL-3 (rIL-3) that rosetted with Staphylococcus aureus increased from 2 +/- 0.5% at 1 week of culture to 7 +/- 2% at 2 weeks, 14 +/- 5% at 3 weeks, and 18 +/- 5% at 4 weeks. Using 125I-labeled IgEPS, the number of IgE receptors per rosetting basophil-like cell was calculated to be 7.3 x 10(4) at 2 weeks, 6.8 x 10(4) at 3 weeks, and 3.9 x 10(4) receptors per rosette positive basophil-like cell at 4 weeks. Finally, the rate of dissociation of IgE from these cultured cells was found to be 4.0 +/- 1.3 x 10(-5) s-1, indicating that IgE bound to IgE receptors on cultured basophil-like cells with high affinity.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1989

IgE receptor-mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells.

Cytoplasts (plasma membrane sacs containing cytoplasm, endoplasmic reticulum, and few organelles) were prepared from rat basophilic leukemia cells by treatment with cytochalasin B and centrifugation at 33 degrees C through stepwise gradients of Ficoll. To compare the relative ability of cytoplasts and cells to generate second-messengers (inositol phosphates, Ca2+) in response to stimulation of the high affinity receptor for IgE, we normalized our results per recovered receptor by using the tightly bound IgE as a marker. This marker correlated well with other estimates of plasma membrane recovery. Furthermore, data normalized on this basis correlated well with data expressed as percentage of phosphoinositides hydrolyzed. The purest fraction of cytoplasts (containing about 6% of the receptors) was satisfactorily devoid of organelles and, at early times, generated about 50% as much inositol phosphates per receptor as did the intact, untreated cells. This response of the cytoplasts, like that of the cells, was totally dependent upon aggregation of the receptors. The response by the cytoplasts (in the 5-min time frame which we examined), unlike that of the cells, was not enhanced by the presence of extracellular Ca2+. Furthermore, unlike the cells, the cytoplasts failed to raise their intracellular free Ca2+ levels after addition of polyvalent Ag. This result suggests that aggregation of the receptors may be insufficient, by itself, to open the normal Ca2+ channels.

Animals↗