bluuebunny/crossref_metadata_2025 huggingface dataset:
The Mouse GATA-2 Gene is Expressed in the Para-Aortic Splanchnopleura and Aorta-Gonads and Mesonephros Region
Authors: Minegishi Naoko , Ohta Jun , Yamagiwa Hironori , Suzuki Norio , Kawauchi Shimako , Zhou Yinghui , Takahashi Satoru , Hayashi Norio , Engel James Douglas , Yamamoto Masayuki
Published: October 2019

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2–expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5′ end of the IS first exon direct expression of β-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5′ end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5′ to the IS exon.
Role of Caspase in a Subset of Human Platelet Activation Responses
Authors: Shcherbina Anna , Remold-O’Donnell Eileen
Published: October 2019

Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.
Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution
Authors: Bayston T.A. , Tripodi A. , Mannucci P.M. , Thompson E. , Ireland H. , Fitches A.C. , Hananeia L. , Olds R.J. , Lane D.A.
Published: October 2019

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.
Hypermethylation of the DAP-Kinase CpG Island Is a Common Alteration in B-Cell Malignancies
Authors: Katzenellenbogen Rachel A. , Baylin Stephen B. , Herman James G.
Published: October 2019

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for γ interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2′-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5′ CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt’s lymphomas and 84% of the B-cell non-Hodgkin’s lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin’s lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase–expressing leukemia cell line, was treated with γ interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt’s lymphoma cell line, only did so when treated with 5-aza-2′-deoxycytidine followed by γ interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of γ interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell–lineage lymphomas.
Fusion of the ets Transcription Factor TEL to Jak2 Results in Constitutive Jak-Stat Signaling
Authors: Ho Jen M.-Y. , Beattie Bryan K. , Squire Jeremy A. , Frank David A. , Barber Dwayne L.
Published: October 2019

To study constitutive Janus kinase signaling, chimeric proteins were generated between the pointed domain of the etstranscription factor TEL and the cytosolic tyrosine kinase Jak2. The effects of these proteins on interleukin-3 (IL-3)–dependent proliferation of the hematopoietic cell line, Ba/F3, were studied. Fusion of TEL to the functional kinase (JH1) domain of Jak2 resulted in conversion of Ba/F3 cells to factor-independence. Importantly, fusion of TEL to the Jak2 pseudokinase (JH2) domain or a kinase-inactive Jak2 JH1 domain had no effect on IL-3–dependent proliferation of Ba/F3 cells. Active TEL-Jak2 constructs (consisting of either Jak2 JH1 or Jak2 JH2+JH1 domain fusions) were constitutively tyrosine-phosphorylated but did not affect phosphorylation of endogeneous Jak1, Jak2, or Jak3. TEL-Jak2 activation resulted in the constitutive tyrosine phosphorylation of Stat1, Stat3, and Stat5 as determined by detection of phosphorylation using activation-specific antibodies and by binding of each protein to a preferential GAS sequence in electrophoretic mobility shift assays. Elucidation of signaling events downstream of TEL-Jak2 activation may provide insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.
Interferon-γ Upregulates CCR5 Expression in Cord and Adult Blood Mononuclear Phagocytes
Authors: Hariharan Deepa , Douglas Steven D. , Lee Benhur , Lai Jian-Ping , Campbell Donald E. , Ho Wen-Zhe
Published: October 2019

The C-C chemokine receptors CCR5 and CCR3 are fusion coreceptors for human immunodeficiency virus (HIV) entry into macrophages. The regulation of their expression influences infectivity by HIV. We report here that interferon-γ (IFN-γ) a cytokine that has bidirectional effects on HIV infection of macrophages, significantly upregulated CCR5 and CCR3 cell surface expression in human mononuclear phagocytes isolated from placental cord blood and adult peripheral blood. Monocytes treated with IFN-γ showed increased chemotaxis to the CCR5 ligands macrophage inflammatory protein-1 (MIP-1) and MIP-1β, confirming the functional relevance of IFN-γ–induced CCR5 expression. However, IFN-γ suppressed HIV entry into macrophages. Interestingly, we demonstrated that IFN-γ inhibited cell surface expression of CD4, the major receptor for HIV. This finding may explain the suppressive effect of IFN-γ on HIV entry into macrophages, despite its enhancing effect on the expression of CCR5 and CCR3 by these cells. In addition, IFN-γ–induced secretion of C-C chemokines (RANTES, MIP-1, and MIP-1β) by mononuclear phagocytes may also suppress HIV entry into macrophages. These data provide further evidence for cytokine-mediated regulation of CCR5 expression and are consistent with a novel paradigm in which cytokines regulate HIV infection and leukocyte migration by reciprocal and opposing effects on the expression of CD4 and chemokine receptors.
Sialylation of the Sialic Acid Binding Lectin Sialoadhesin Regulates Its Ability to Mediate Cell Adhesion
Authors: Barnes Yvonne C. , Skelton Tim P. , Stamenkovic Ivan , Sgroi Dennis C.
Published: October 2019

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3–linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.
Increased Sensitivity of Acute Myeloid Leukemias to Lovastatin-Induced Apoptosis: A Potential Therapeutic Approach
Authors: Dimitroulakos Jim , Nohynek Dana , Backway Karen L. , Hedley David W. , Yeger Herman , Freedman Melvin H. , Minden Mark D. , Penn Linda Z.
Published: October 2019

We recently demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of de novo cholesterol synthesis, was a potential mediator of the biological effects of retinoic acid on human neuroblastoma cells. The HMG-CoA reductase inhibitor, lovastatin, which is used extensively in the treatment of hypercholesterolemia, induced a potent apoptotic response in human neuroblastoma cells. This apoptotic response was triggered at lower concentrations and occurred more rapidly than had been previously reported in other tumor-derived cell lines, including breast and prostate carcinomas. Because of the increased sensitivity of neuroblastoma cells to lovastatin-induced apoptosis, we examined the effect of this agent on a variety of tumor cells, including leukemic cell lines and primary patient samples. Based on a variety of cytotoxicity and apoptosis assays, the 6 acute lymphocytic leukemia cell lines tested displayed a weak apoptotic response to lovastatin. In contrast, the majority of the acute myeloid leukemic cell lines (6/7) and primary cell cultures (13/22) showed significant sensitivity to lovastatin-induced apoptosis, similar to the neuroblastoma cell response. Of significance, in the acute myeloid leukemia, but not the acute lymphocytic leukemia cell lines, lovastatin-induced cytotoxicity was pronounced even at the physiological relevant concentrations of this agent. Therefore, our study suggests the evaluation of HMG-CoA reductase inhibitors as a therapeutic approach in the treatment of acute myeloid leukemia.
Fusion of ETV6 to Neurotrophin-3 Receptor TRKC in Acute Myeloid Leukemia With t(12;15)(p13;q25)
Authors: Eguchi Mariko , Eguchi-Ishimae Minenori , Tojo Arinobu , Morishita Kazuhiro , Suzuki Katsuyuki , Sato Yuko , Kudoh Shiori , Tanaka Kimio , Setoyama Misao , Nagamura Fumitaka , Asano Shigetaka , Kamada Nanao
Published: October 2019

Chromosome translocations involving band 12p13 are known to be involved in a variety of hematologic malignancies, some of them resulting in rearrangement of the ETV6/TEL gene. Applying the fluorescence in situ hybridization (FISH) method, we found a cryptic translocation t(12;15)(p13;q25) in an adult acute myeloid leukemia (AML) patient. Hybridization with cosmid probes showed that the ETV6 gene was rearranged in this translocation. A patient-specific cDNA library was screened with ETV6 cDNA, and a novel fusion transcript was identified between the ETV6 andTRKC/NTRK3 gene located on 15q25. TRKC is a receptor tyrosine kinase that is activated by neurotrophin-3 (NT-3). It is known to be expressed broadly in neural tissues but not in hematologic cells, so far. ETV6-TRKC chimeric transcript encoded the pointed (PNT) domain of the ETV6 gene that fused to the protein-tyrosine kinase (PTK) domain of the TRKC gene. Two types of fusion transcript were determined, one that included the entire PTK domain of TRKC and the other in which the 3′-terminal 462 bp of TRKC was truncated within the PTK domain. Western blot analysis showed the expression of both chimeric proteins of 52 and 38 kD in size. Our results suggest that chimeric PTK expressed in the leukemic cells may contribute to cellular transformation by abnormally activating TRK signaling pathways. Moreover, this is the first report on truncated neurotrophin receptors associated in leukemia.
Activation of Nitric Oxide Release and Oxidative Metabolism by Leukotrienes B4, C4, and D4 in Human Polymorphonuclear Leukocytes
Authors: Lärfars Gerd , Lantoine Frédérique , Devynck Marie-Aude , Palmblad Jan , Gyllenhammar Hans
Published: October 2019

Because arachidonate metabolites are potent mediators of inflammation, we have studied the effects of leukotriene B4(LTB4) and the cysteinyl leukotrienes C4 and D4 (LTC4 and LTD4) on the release of nitric oxide (NO), in vitro, by human polymorphonuclear granulocytes (PMN). Two independent and highly sensitive real-time methods were used for these studies, ie, the NO-dependent oxidation of oxyhemoglobin (HbO2) to methemoglobin and a NO-sensitive microelectrode. When activated with LTB4, LTC4, or LTD4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S,12S-diHETE, PMN produced NO in a stimulus- and concentration-dependent manner. The rank order of potency was LTB4 = LTC4 > LTD4, corresponding to 232 ± 50 pmol of NO/106 PMN for 100 nmol/L LTB4 after 30 minutes. The kinetic properties of the responses were similar for all three leukotrienes with a maximum response at 13 ± 3 minutes. Cysteinyl leukotriene and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with Bordetella pertussis toxin, or chelation of cytosolic Ca2+, [Ca2+]i, also efficiently inhibited this response. In contrast, treatment of PMN with cytochalasin B (5 μg/mL) enhanced the LTB4-induced NO formation by 86%. Thus, this is the first demonstration that the cysteinyl leukotrienes LTC4 and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i-dependent mechanisms. This effect differs from activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, for which only LTB4is an activator.
Quantification of T-Cell Progenitors During Ontogeny: Thymus Colonization Depends on Blood Delivery of Progenitors
Authors: Dunon D. , Allioli N. , Vainio O. , Ody C. , Imhof B.A.
Published: October 2019

An in vivo thymus reconstitution assay based on intrathymic injection of hematopoietic progenitors into irradiated chicks was used to determine the number of T-cell progenitors in peripheral blood, paraaortic foci, bone marrow (BM), and spleen during ontogeny. This study allowed us to analyze the regulation of thymus colonization occurring in three waves during embryogenesis. It confirmed that progenitors of the first wave of thymus colonization originate from the paraaortic foci, whereas progenitors of the second and the third waves originate from the BM. The analysis of the number of T-cell progenitors indicates that each wave of thymus colonization is correlated with a peak number of T-cell progenitors in peripheral blood, whereas they are almost absent during the periods defined as refractory for colonization. Moreover, injection of T-cell progenitors into the blood circulation showed that they homed into the thymus without delay during the refractory periods. Thus, thymus colonization kinetics depend mainly on the blood delivery of T-cell progenitors during embryogenesis.
Antifactor VIII Antibody Inhibiting Allogeneic but not Autologous Factor VIII in Patients With Mild Hemophilia A
Authors: Peerlinck Kathelijne , Jacquemin Marc G. , Arnout Jef , Hoylaerts Marc F. , Gilles Jean Guy G. , Lavend’homme Renaud , Johnson Karen M. , Freson Kathleen , Scandella Dorothea , Saint-Remy Jean-Marie R. , Vermylen Jos
Published: October 2019

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.
Stomatocytosis Is Absent in “Stomatin”-Deficient Murine Red Blood Cells
Authors: Zhu Yiwen , Paszty Chris , Turetsky Tikva , Tsai Susan , Kuypers Frans A. , Lee Gloria , Cooper Philip , Gallagher Patrick G. , Stevens Mary E. , Rubin Edward , Mohandas Narla , Mentzer William C.
Published: October 2019

To examine the relationship between erythrocyte membrane protein 7.2b deficiency and the hemolytic anemia of human hereditary stomatocytosis, we created 7.2b knock-out mice by standard gene targeting approaches. Immunoblots showed that homozygous knock-out mice completely lacked erythrocyte protein 7.2b. Despite the absence of protein 7.2b, there was no hemolytic anemia and mouse red blood cells (RBCs) were normal in morphology, cell indices, hydration status, monovalent cation content, and ability to translocate lipids. The absence of the phenotype of hereditary stomatocytosis implies that protein 7.2b deficiency plays no direct role in the etiology of this disorder and casts doubt on the previously proposed role of this protein as a mediator of cation transport in RBC.
A CD4-Independent Interaction of Human Immunodeficiency Virus-1 gp120 With CXCR4 Induces Their Cointernalization, Cell Signaling, and T-Cell Chemotaxis
Authors: Missé Dorothée , Cerutti Martine , Noraz Nelly , Jourdan Patrick , Favero Jean , Devauchelle Gérard , Yssel Hans , Taylor Naomi , Veas Francisco
Published: October 2019

The gp120 envelope glycoprotein of human immunodeficiency virus-1 (HIV-1) interacts with the CXCR4 chemokine receptor, but it is not known whether gp120 activates CXCR4-mediated signaling cascades in the same manner as its natural ligand, SDF1. We assessed the effects of wild-type gp120 and a mutant gp120 that interacts with CXCR4 but not CD4 on CD4−/CXCR4+ cells and CD4+/CXCR4+ cells, respectively. Under both experimental conditions, the interaction of CXCR4 and gp120 resulted in their CD4-independent cointernalization. Both molecules were translocated into early endosomes, whereas neither protein could be detected in late endosomes. Binding of gp120 to CXCR4 resulted in a CD4-independent phosphorylation of Pyk2 and an induction of chemotactic activity, demonstrating that this interaction has functional consequences. Interestingly, however, whereas SDF1 activated the ERK/MAP kinase pathway, this cascade was not induced by gp120. Together, these results suggest that the pathology of HIV-1 infection may be modulated by the distinct signal transduction pathway mediated by gp120 upon its interaction with CXCR4.
Neutrophil Activation and Hemostatic Changes in Healthy Donors Receiving Granulocyte Colony-Stimulating Factor
Authors: Falanga A. , Marchetti M. , Evangelista V. , Manarini S. , Oldani E. , Giovanelli S. , Galbusera M. , Cerletti C. , Barbui T.
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 μg/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF–treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.
Human Platelets Contain SNARE Proteins and a Sec1p Homologue That Interacts With Syntaxin 4 and Is Phosphorylated After Thrombin Activation: Implications for Platelet Secretion
Authors: Reed Guy L. , Houng Aiilyan K. , Fitzgerald Michael L.
Published: October 2019

In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins—VAMP, SNAP-25, and syntaxin 4—that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 ± 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.
Increased BAX Expression Is Associated With an Increased Risk of Relapse in Childhood Acute Lymphocytic Leukemia
Authors: Hogarth Linda A. , Hall Andrew G.
Published: October 2019

Studies in cell lines have indicated that expression of the BCL-2 family of proteins is an important determinant of chemotherapy-induced apoptosis; however, the level of expression of these proteins in childhood acute lymphoblastic leukemia (ALL) has not been extensively reported. Using quantitative Western blotting we have determined the level of expression of BCL-2, BAX, MCL-1, and BCL-X in lymphoblasts from 47 children with ALL (33 at presentation only, 4 at relapse only, and 10 at both presentation and on relapse). Results were determined as a ratio to actin as an internal control. BCL-2, BAX, and MCL-1 were detected in all samples. BCL-XL was only detected in 6 cases (4 at presentation and 2 at relapse) and BCL-XS in none. No correlation was found between expression and white blood cell count, age at diagnosis, gender, or blast karyotype. BCL-2 levels and the BCL/BAX and MCL-1/BAX ratios were found to be significantly higher in B-lineage as compared with T-lineage disease (P < .003, .02, and .02, respectively). No consistent pattern of change in expression was noted in the 10 cases studied at both presentation and relapse. Kaplan-Meier analysis showed a significant correlation between high BAX expression and an increased probability of relapse (P< .05 by the log rank test), suggesting that chemosensitivity in leukemic blasts may be regulated by factors that override the BCL-2 pathway.
A Novel SH2-Containing Phosphatidylinositol 3,4,5-Trisphosphate 5-Phosphatase (SHIP2) Is Constitutively Tyrosine Phosphorylated and Associated With src Homologous and Collagen Gene (SHC) in Chronic Myelogenous Leukemia Progenitor Cells
Authors: Wisniewski David , Strife Annabel , Swendeman Steve , Erdjument-Bromage Hediye , Geromanos Scott , Kavanaugh W. Michael , Tempst Paul , Clarkson Bayard
Published: October 2019

Because of the probable causal relationship between constitutive p210bcr/abl protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210bcr/abl-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain–containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210bcr/abl-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4,5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210bcr/abl-mediated myeloid expansion.
Human Signal-Regulatory Protein Is Expressed on Normal, But Not on Subsets of Leukemic Myeloid Cells and Mediates Cellular Adhesion Involving Its Counterreceptor CD47
Authors: Seiffert Martina , Cant Charles , Chen Zhengjun , Rappold Irene , Brugger Wolfram , Kanz Lothar , Brown Eric J. , Ullrich Axel , Bühring Hans-Jörg
Published: October 2019

Signal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. To analyze the expression and function of SIRPs, we prepared soluble recombinant fusion proteins of the extracellular regions of SIRP1 and SIRP2, as well as a variety of monoclonal antibodies (MoAbs) against these domains. The antibodies reacted predominantly with monocytes, granulocytes, dendritic cells, and their precursors, as well as with bone marrow CD34+, AC133+, CD90+hematopoietic stem/progenitor cells. In contrast, SIRP expression was absent or significantly reduced on the majority of myeloid blasts from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). Functional studies showed that the extracellular domains of SIRP1 and SIRP2 support adhesion of a number of primary hematopoietic cells and cell lines. This interaction could be blocked by 4 of 7 SIRP1-reactive MoAbs. In addition, SIRP1 and SIRP2 competed for the same cell binding site, suggesting a common widely expressed SIRP ligand. In an approach to identify this molecule, MoAbs were generated against the SIRP-binding cell line CCRF-CEM, and MoAb CC2C6 was selected because of its capacity to inhibit cell binding to SIRP1. Further analysis showed that this antibody recognized CD47, a ubiquitously expressed plasma membrane protein previously implicated in integrin function, host defense action, and neutrophil migration. In this study, we identify CD47 as the extracellular ligand for human SIRP and show that these two counterreceptors are involved in cellular adhesion.
Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells
Authors: Burger Jan A. , Burger Meike , Kipps Thomas J.
Published: October 2019

Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.
Lipoprotein (a) and Genetic Polymorphisms of Clotting Factor V, Prothrombin, and Methylenetetrahydrofolate Reductase Are Risk Factors of Spontaneous Ischemic Stroke in Childhood
Authors: Nowak-Göttl Ulrike , Sträter Ronald , Heinecke Achim , Junker Ralf , Koch Hans-Georg , Schuierer Gerhard , von Eckardstein Arnold
Published: October 2019

Ischemic stroke is a rare event in childhood. In approximately one third of cases no obvious underlying cause or disorder can be detected. We investigated the importance of genetic risk factors of venous thromboembolism in childhood or stroke in adulthood as risk factors for spontaneous ischemic stroke in children. One hundred forty-eight Caucasian infants and children (aged 0.5 to 16 years) with stroke and 296 age-matched controls from the same geographic areas as the patients were analyzed for increased lipoprotein (a) [Lp(a)] levels >30 mg/dL; for the presence of the factor V (FV) G1691A mutation, the prothrombin (PT) G20210A variant, and the TT677 genotype of methylenetetrahydrofolate reductase (MTHFR); and deficiencies of protein C, protein S, and antithrombin. The following frequencies (patients v controls), odds ratios (ORs), and confidence intervals (CIs) of single risk factors were found: Lp(a) >30 mg/dL (26.4% v 4.7%; OR/CI, 7.2/3.8 to 13.8; P < .0001), FV G1691A (20.2% v 4%; OR/CI, 6/2.97 to 12.1; P < .0001), protein C deficiency (6% v 0.67%; OR/CI, 9.5/2 to 44.6; P = .001), PT G20210A (6% v 1.3%; OR/CI, 4.7/1.4 to 15.6; P = .01), and the MTHFR TT677 genotype (23.6% v 10.4%; OR/CI, 2.4/1.53 to 4.5; P < .0001). A combination of the heterozygous FV G1691A mutation with increased Lp(a) (n = 11) or the MTHFR TT677 genotype (n = 5) was found in 10.8% of cases, but only 0.3% of controls (OR/CI, 35.75/4.7 to 272;P < .0001). Increased Lp (a) levels, the FV G1691A mutation, protein C deficiency, the prothrombin G20210A variant, and the MTHFR TT677 are important risk factors for spontaneous ischemic stroke in childhood.
Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules
Authors: Heijnen Harry F.G. , Schiel Anja E. , Fijnheer Rob , Geuze Hans J. , Sixma Jan J.
Published: October 2019

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.
Inhibition of Caspase Cascade by HTLV-I Tax Through Induction of NF-κB Nuclear Translocation
Authors: Kawakami Atsushi , Nakashima Tomoki , Sakai Hideaki , Urayama Satoshi , Yamasaki Satoshi , Hida Ayumi , Tsuboi Masahiko , Nakamura Hideki , Ida Hiroaki , Migita Kiyoshi , Kawabe Yojiro , Eguchi Katsumi
Published: October 2019

NF-κB is required for prevention of apoptosis. We examined the importance of human T-cell leukemia virus–I (HTLV-I) Tax protein to stimulate NF-κB nuclear translocation, thus preventing apoptosis. Jurkat cells and JPX-9 cells in which the inducible Tax expression plasmid vector was stably transfected were used in the present study. Both Jurkat and Tax− JPX-9 cells had small amounts of basal nuclear NF-κB activity. The addition of NF-κB inhibitors suppressed NF-κB nuclear translocation of the cells, thus inducing apoptosis. Sequential activation of caspases from caspase-8 to caspase-3 was shown during this process. NF-κB nuclear translocation in JPX-9 cells was stimulated through Tax expression, and both the activation of caspases and apoptosis induced by NF-κB inhibitors were significantly suppressed in the Tax+ JPX-9 cells. The expression of Bcl-2, Bax, and Bcl-x was not changed among Jurkat, Tax− JPX-9, and Tax+ JPX-9 cells in the presence or absence of NF-κB inhibitors. X-chromosome–linked inhibitor of apoptosis (XIAP) protein expression in Tax−JPX-9 cells was significantly suppressed by NF-κB inhibitors, however, its expression in Tax+ JPX-9 cells was maintained even by the addition of NF-κB inhibitors. Our results suggest that the activation of NF-κB via Tax protein in HTLV-I infected cells renders the cells resistant to apoptosis. The expression of anti-apoptotic gene products such as XIAP to suppress caspase cascade, results in an increase of cytokine production and cell proliferation; one of the proposed mechanisms that promotes autoimmune disorders such as Sjögren’s syndrome and rheumatoid arthritis found in HTLV-I seropositive subjects.
Granulocyte-Macrophage Colony-Stimulating Factor Upregulates Reduced 5-Lipoxygenase Metabolism in Peripheral Blood Monocytes and Neutrophils in Acquired Immunodeficiency Syndrome
Authors: Coffey Michael J. , Phare Susan M. , Cinti Sandro , Peters-Golden Marc , Kazanjian Powel H.
Published: October 2019

Leukotrienes (LT) are mediators derived from the 5-lipoxygenase (5-LO) pathway, which play a role in host defense, and are synthesized by both monocytes (peripheral blood monocyte [PBM]) and neutrophils (PMN). Because 5-LO metabolism is reduced in alveolar macrophages and PMN from acquired immunodeficiency syndrome (AIDS) subjects, we investigated the synthesis of LT by PBM and PMN from these subjects. There was a reduction (74.2% ± 8.8% of control) in LT synthesis in PBM from human immunodeficiency virus (HIV)-infected compared with normal subjects. Expression of 5-LO (51.2% ± 8.8% of control), and 5-LO activating protein (FLAP) (48.5% ± 8.0% of control) was reduced in parallel. We hypothesized that this reduction in LT synthetic capacity in PBM and PMN was due to reduced cytokine production by CD4 T cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We treated 10 AIDS subjects with GM-CSF for 5 days. PBM 5-LO metabolism ex vivo was selectively increased after GM-CSF therapy and was associated with increased 5-LO and FLAP expression. PMN leukotriene B4(LTB4) synthesis was also augmented and associated with increased 5-LO, FLAP, and cytosolic phospholipase A2 expression. In conclusion, as previously demonstrated for PMN, PBM from AIDS subjects also demonstrate reduced 5-LO metabolism. GM-CSF therapy reversed this defect in both PBM and PMN. In view of the role of LT in antimicrobial function, cytokine administration in AIDS may play a role as adjunct therapy for infections.
HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells
Authors: Riedel H.D. , Muckenthaler M.U. , Gehrke S.G. , Mohr I. , Brennan K. , Herrmann T. , Fitscher B.A. , Hentze M.W. , Stremmel W.
Published: October 2019

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.
Distinct Roles of JNKs/p38 MAP Kinase and ERKs in Apoptosis and Survival of HCD-57 Cells Induced by Withdrawal or Addition of Erythropoietin
Authors: Shan Rujiao , Price James O. , Gaarde William A. , Monia Brett P. , Krantz Sanford B. , Zhao Zhizhuang Joe
Published: October 2019

Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.
Glycoprotein V-Deficient Platelets Have Undiminished Thrombin Responsiveness and Do Not Exhibit a Bernard-Soulier Phenotype
Authors: Kahn Mark L. , Diacovo Thomas G. , Bainton Dorothy F. , Lanza Francois , Trejo JoAnn , Coughlin Shaun R.
Published: October 2019

Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.