bluuebunny/crossref_metadata_2025_split huggingface dataset:
Signal transduction of the human granulocyte-macrophage colony- stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins
Authors: Kanakura Y , Druker B , Cannistra SA , Furukawa Y , Torimoto Y , Griffin JD 
Published: October 2019

Abstract
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine- kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM- CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM- CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.
Active site-specific immunoassays
Authors: Mann KG , Williams EB , Krishnaswamy S , Church W , Giles A , Tracy RP 
Published: October 2019

Abstract
This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.
Comparative levels of CALLA/neutral endopeptidase on normal granulocytes, leukemic cells, and transfected COS-1 cells
Authors: Tran-Paterson R , Boileau G , Giguere V , Letarte M 
Published: October 2019

Abstract
We discovered that the common acute lymphoblastic leukemia antigen, CALLA (CD10), was identical to human neutral endopeptidase 3.4.24.11 (NEP), a Zn-binding glycoprotein with an extracellular active site capable of hydrolyzing several biologically active peptides. In this study we compare the expression of CALLA/NEP in terms of antigenic density and enzymatic activity at the cell surface and of messenger RNA (mRNA) levels on granulocytes, leukemic cells, and CALLA-transfected COS-1 cells. Mature granulocytes, the only readily available source of normal human CALLA, express relatively low but constant levels of antigen, NEP activity (3.5 pmol/min/10(6) cells), and mRNA. The two major CALLA-mRNA species of 6.5 kb and 3.8 kb, observed to date in a variety of cells and tissues, were also found in four independent granulocyte preparations. With leukemia cell lines, a correlation was established between the density of CALLA antigen and the level of enzymatic activity (3.4 to 21.0 pmol/min/10(6) cells). This paper constitutes the first report of NEP activity on blast cells derived from patients with non-T acute lymphoblastic leukemia (ALL); the levels of activity were variable (1.5 to 35.9 pmol/min/10(6) cells for six cases) but correlated with the level of CALLA assessed by flow cytometry. Heterogeneous levels of expression of the CALLA-mRNA species were also observed in non-T ALL cases that correlated with the level of CALLA expression at the surface of these cells. Very high levels of NEP activity were achieved by transfecting COS-1 cells with pSV-CALLA; 20% of the transfected cells were CALLA+ and expressed 550 pmol/min/10(6) cells. Extracts prepared from COS-1 cells transfected with pSV-CALLA (carrying human NEP cDNA) and pSVENK19 (carrying rabbit NEP-cDNA), respectively, gave Michaelis constant (Km) values of 50 mumol/L and similar inhibition curves with thiorphan. Thus the recombinant proteins encoded by these two genes have similar enzymatic properties, confirming the high degree of their structural relatedness. The expression of high levels of CALLA/NEP on COS-1 cells should allow the use of this system to test the effects of specific mutations on activity and might lead to the understanding of the role of CALLA in the onset and/or progression of leukemia.
RFLP and deletion analysis for X-linked chronic granulomatous disease using the cDNA probe: potential for improved prenatal diagnosis and carrier determination
Authors: Pelham A , O'Reilly MA , Malcolm S , Levinsky RJ , Kinnon C 
Published: October 2019

Abstract
The molecular basis of X-linked chronic granulomatous disease (X-CGD) has recently been elucidated and the defective gene identified and isolated. Two restriction fragment-length polymorphisms have been identified using the X-CGD cDNA probe. We have analyzed eight families with X-CGD and seven normal, unrelated females and have demonstrated that these polymorphisms are not in linkage disequilibrium. This should increase to approximately 50% the proportion of families to whom first- trimester prenatal diagnosis can be offered. Unambiguous determination of carrier status in related females in informative families will also be possible. In addition, we have identified an apparently unique small deletion in the X-CGD gene in a family affected by this disease, members of which are not informative for either polymorphism. This will allow prenatal diagnosis and carrier determination in this family.
Relationship of the human erythrocyte Wrb antigen to an interaction between glycophorin A and band 3
Authors: Telen MJ , Chasis JA 
Published: October 2019

Abstract
The Wrb antigen is a high-frequency human erythrocyte antigen invariably absent from En (a-) erythrocytes, which lack glycophorin A. However, glycophorin A from En (a+) Wr (a+b-) red cells has an amino acid sequence identical to that of glycophorin A from Wr (b+) erythrocytes. Evidence has suggested that the Wrb antigen may require the interaction of glycophorin A with either a lipid moiety or with another erythrocyte-integral membrane protein, band 3. We have investigated the role of band 3 in Wrb expression using murine monoclonal antibodies (MoAbs) with Wrb specificity. These antibodies reacted by radioimmunoassay (RIA) only with cells expressing both glycophorin A and band 3. In immunoprecipitation studies, Wrb antibodies immunoprecipitated both band 3 and glycophorin A, while antibodies specific for band 3 or glycophorin precipitated only the protein with which they were reactive. These data strongly suggest that band 3 is the other membrane component necessary for expression of Wrb and that band 3 and glycophorin A are closely associated in the erythrocyte membrane.
Oxidation-induced changes in microrheologic properties of the red blood cell membrane
Authors: Hebbel RP , Leung A , Mohandas N 
Published: October 2019

Abstract
It has been hypothesized that some of the irreversible microrheologic abnormalities of sickle red blood cell (RBC) membranes could result from autoxidative perturbation. To model this possibility, we used micromechanical manipulation to examine the static extensional rigidity and inelastic or plastic behavior of normal RBCs exposed to phenazine methosulfate (PMS), an agent that generates superoxide from within the cell. In response to this stress, RBC membranes became stiff as evidenced by increasing extensional rigidity. At 50 mumol/L PMS they were as stiff as the membranes of most dense, dehydrated sickle RBCs; and at 25 mumols/L PMS the membranes were similar to somewhat less dense sickle RBCs. When examined for inelastic behavior, RBCs exposed to PMS even at 10 mumols/L showed hysteresis in loading and unloading phases of the curve relating aspiration length to suction pressure, and they developed membrane bumps that persisted after RBC release from the pipette. Examination of single cells in both isotonic and hypotonic buffers showed that the effect of PMS on RBC microheology is not mediated by cellular dehydration. Independent confirmation of the membrane stiffening effect of PMS was obtained by ektacytometric analysis of resealed RBC ghosts, with sickle-like increases in membrane rigidity observed between 50 and 100 mumol/L PMS. The rigidity of these ghosts was partially ameliorated by exposure to a thiol reductant. In terms of biochemical abnormalities, treated RBCs became significantly different from control RBCs at 25 mumol/L PMS, at which point they just began to enter the sickle range for amounts of membrane thiol oxidation and membrane-associated heme. The sickle average was achieved at 50 mumol/L PMS (for thiol oxidation) to 100 mumol/L PMS (for membrane heme). Thus, micromolar concentrations of PMS induce abnormalities of membrane microrheology that closely mimic those of unmanipulated sickle RBCs while reproducing similar degrees of oxidative biochemical change. We conclude that membrane protein oxidation could explain existence of an irreversible component to the abnormal rheology of the sickle membrane.
2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S
Authors: Poillon WN , Kim BC 
Published: October 2019

Abstract
We have established that 2,3-diphosphoglycerate (2,3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (csat) of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic csat assay system, a steep dependence of csat on pH in the physiologic range 7.0 to 7.6 was shown for both stripped (Hb) and DPG-saturated deoxy-Hb S (Hb-DPG). The solubility-pH profile for Hb under near-physiologic buffer conditions also showed that csat increased steeply in the same pH range (6.8 to 7.6). The effect of 2,3- DPG on csat under near-physiologic conditions was evaluated separately. At pH 7.20, the pH of the human red blood cell, csat values for Hb and Hb-DPG were 19.56 +/- 0.14 and 17.95 +/- 0.45 g/dL, respectively, indicating that the solubility of Hb-DPG is lower than that of Hb by 8.2% +/- 2.3%. Thus, binding of 2,3-DPG in the beta-cleft promotes the polymerization of deoxy-Hb S, the ultimate determinant of cell sickling. Furthermore, because of the abnormal Bohr effect of sickle blood (approximately double that of normal blood), the intracellular pH of deoxygenated sickle erythrocytes should be approximately 0.28 pH unit higher than that of oxygenated cells (ie, 7.41 v 7.13). At the higher pH, the corresponding csat for Hb-DPG is 20.22 g/dL, which is the best estimate of the intrinsic solubility of T-state Hb S under conditions that approximate closely those of pH, temperature, ionic strength, and 2,3-DPG saturation in the fully desaturated sickle erythrocyte.
Growth and detection of human bone marrow B-lineage colonies
Authors: McGinnes K , Keystone E , Bogoch E , Hastings D , Messner HA , Jamal N , Paige CJ 
Published: October 2019

Abstract
A reproducible method for growing normal human bone marrow B-lineage colonies in agar is described. The clonogenic cells require a rich medium, Opti-MEM (GIBCO/BRL, Burlington, Ontario, Canada), and a source of T-cell-derived factors for growth. The conditions described appear to be limiting for the colony progenitor, suggesting assay clonality. Three novel methods that permit routine and rapid detection of these human B-cell colonies are also described. Colonies containing cells that secrete immunoglobulin are detected by plaquing and protein immunoblotting, while RNA transcripts can be detected by RNA colony blotting. The detection of more than one secreted immunoglobulin isotype or RNA species in a single colony can also be achieved. This B- cell colony assay and the associated detection methods will allow the further delineation of human B lymphopoiesis in both normal and disease states.
T-cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro
Authors: Williams DE , Morrissey PJ , Mochizuki DY , de Vries P , Anderson D , Cosman D , Boswell HS , Cooper S , Grabstein KH , Broxmeyer HE 
Published: October 2019

Abstract
T-cell growth factor P40 was examined for possible effects on murine interleukin-3 (IL-3)-dependent myeloid cell lines and freshly isolated murine bone marrow cells. The results showed that P40 stimulated the proliferation of some IL-3-dependent myeloid cell lines of both early myeloid and mast cell phenotype and synergized with IL-3. P40 did not promote proliferation of fresh bone marrow cells, bone marrow enriched for early myeloid cells by 5-fluorouracil treatment, or bone marrow derived mast cells as assessed in 3H-TdR incorporation assays. P40 did not influence the growth of murine colony-forming unit granulocyte- macrophage in agar cultures, either alone or in the presence of optimal or sub-optimal concentrations of CSF-1, GM-colony-stimulating factor, or IL-3. P40 did potentiate burst-forming unit-erythroid (BFU-E) formation in the presence of erythropoietin; however, this was dependent on the cell plating density, suggesting an indirect stimulation of BFU-E by P40. The indirect nature of P40 action on BFU-E was further demonstrated in cell separation experiments and indicated that the effect was mediated by T cells. These data expand the repertoire of cells that P40 influences.
Fish oil: a potent inhibitor of platelet adhesiveness [see comments]
Authors: Li XL , Steiner M 
Published: October 2019

Abstract
The effect of fish oil administration on platelet function was studied in eight normal individuals, four men and four women, who received fish oil equivalent to 6 g eicosapentaenoic acid per day for a period of 25 days. Platelet aggregation, platelet adhesion, phospholipid and fatty acid distribution were measured at periodic intervals before, during, and after the period of fish oil administration. Platelet aggregation induced by arachidonic acid, adenosine diphosphate, and collagen showed a moderate increase in ED 50 in response to the administration of fish oil. Conversely, platelet adhesion to fibrinogen and collagen I, which was studied at low shear rates in a laminar flow chamber, showed a striking 60% to 65% decrease after fish oil supplementation of the diet. The change in adhesiveness could be correlated with the pseudopodia formed in response to agonistic stimulation. Scanning electron microscopic examination of adherent platelets showed an overall reduction of pseudopodia that appeared short and stubby on fish oil administration. The profile of the fatty acids extracted from plasma confirmed compliance of the volunteers with their dietary supplements. Analysis of phospholipids showed changes in sphingomyelin, lysophosphatidylcholine, and phosphatidylcholine between pseudopodia and platelet cell bodies. Fish oil administration did not affect their overall distribution except for a moderate decrease in phosphatidylethanolamine in platelet pseudopodia. Changes were also recognized in the total fatty acids extracted from platelets, affecting primarily arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. There were no changes in platelet adhesiveness in a group of five normal individuals who received a vegetable oil supplement of equal dose and duration as that of the fish oil. We conclude from these studies that fish oil, at least when administered over a limited period of time, is an effective inhibitor of platelet adhesiveness.
Sulfation of tyrosine residues in coagulation factor V
Authors: Hortin GL 
Published: October 2019

Abstract
Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.
Kaposi‧s sarcoma in human T-cell leukemia virus type I-associated adult T-cell leukemia
Authors: Greenberg SJ , Jaffe ES , Ehrlich GD , Korman NJ , Poiesz BJ , Waldmann TA 
Published: October 2019

Abstract
Kaposi's sarcoma (KS) developed in a patient with human T-cell leukemia virus type I (HTLV-I)-associated adult T-cell leukemia who was treated with a short-term course of monoclonal antibody immunotherapy. The presentation was transient and temporally related to the underlying clinical course. The association of KS in an HTLV-I infected, but not human immunodeficiency virus (HIV)-infected, individual should alert investigators to the occurrence of KS in retroviral-associated diseases other than acquired immunodeficiency disease syndrome. Recognition of the similarities and differences between HTLV-I and HIV infections may provide insights concerning the angiopathogenesis of KS.
Isoquinolinesulfonamide protein kinase inhibitors H7 and H8 enhance the effects of granulocyte-macrophage colony-stimulating factor (GM-CSE) on neutrophil function and inhibit GM-CSF receptor internalization
Authors: Khwaja A , Roberts PJ , Jones HM , Yong K , Jaswon MS , Linch DC 
Published: October 2019

Abstract
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) increases neutrophil surface expression of the cellular adhesion molecule CD11b and primes the respiratory burst stimulated by the bacterial peptide f-met-leuphe (FMLP). We have examined the effects of the isoquinolinesulfonamide protein kinase inhibitors H7 and H8 on these functions of GM-CSF using whole blood assays. Concentrations of H7 and H8 that inhibited the 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated upregulation of CD11b expression and activation of the respiratory burst, both augmented the effects of GM-CSF. H7 and H8 enhanced the GM-CSF-stimulated increase in CD11b expression to 215% +/- 10% (P less than .05) and 233% +/- 45% (P less than .05), respectively, of the value obtained with GM-CSF alone. The GM-CSF priming of the FMLP- stimulated oxidative burst was increased to 190% +/- 44% (P less than .01) by preincubation with H7 and to 172% +/- 25% (P less than .01) with H8. Preincubation with H8 did not affect overall binding of 125I- GM-CSF to neutrophils, but inhibited GM-CSF receptor internalization after ligand binding (P less than .05). These data indicate that the effects of GM-CSF are not mediated by protein kinase C and that a phosphorylation event down-modulates the neutrophil response to GM-CSF. It suggests that internalization of the receptor-ligand complex is not a rate-limiting step in signal transduction, and that regulation of the rate of internalization may be an important level of control of the activity of GM-CSF.