bluuebunny/crossref_metadata_2025_split huggingface dataset:
Consequences of GATA-1 Deficiency in Megakaryocytes and Platelets
Authors: Vyas Paresh , Ault Kenneth , Jackson Carl W. , Orkin Stuart H. , Shivdasani Ramesh A. 
Published: October 2019

AbstractIn the absence of the hematopoietic transcription factor GATA-1, mice develop thrombocytopenia and an increased number of megakaryocytes characterized by marked ultrastructural abnormalities. These observations establish a critical role for GATA-1 in megakaryopoiesis and raise the question as to how GATA-1 influences megakaryocyte maturation and platelet production. To begin to address this, we have performed a more detailed examination of the megakaryocytes and platelets produced in mice that lack GATA-1 in this lineage. Our analysis demonstrates that compared with their normal counterparts, GATA-1–deficient primary megakaryocytes exhibit significant hyperproliferation in liquid culture, suggesting that the megakaryocytosis seen in animals is nonreactive. Morphologically, these mutant megakaryocytes are small and show evidence of retarded nuclear and cytoplasmic development. A significant proportion of these cells do not undergo endomitosis and express markedly lower levels of mRNA of all megakaryocyte-associated genes tested, including GPIb, GPIbβ, platelet factor 4 (PF4), c-mpl, and p45 NF-E2. These results are consistent with regulation of a program of megakaryocytic differentiation by GATA-1. Bleeding times are significantly prolonged in mutant animals. GATA-1–deficient platelets show abnormal ultrastructure, reminiscent of the megakaryocytes from which they are derived, and exhibit modest but selective defects in platelet activation in response to thrombin or to the combination of adenosine diphosphate (ADP) and epinephrine. Our findings indicate that GATA-1 serves multiple functions in megakaryocyte development, influencing both cellular growth and maturation.
Expression of a Functional N-Methyl-d-Aspartate–Type Glutamate Receptor by Bone Marrow Megakaryocytes
Authors: Genever Paul G. , Wilkinson David J.P. , Patton Amanda J. , Peet Nicky M. , Hong Ying , Mathur Anthony , Erusalimsky Jorge D. , Skerry Tim M. 
Published: October 2019

AbstractBetter understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of theN-methyl-d-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.
Glycoprotein IIb-IIIa Is Expressed on Avian Multilineage Hematopoietic Progenitor Cells
Authors: Ody Christiane , Vaigot Pierre , Quéré Pascale , Imhof Beat A. , Corbel Catherine 
Published: October 2019

AbstractThe fibrinogen receptor GPIIb-IIIa integrin is known to be expressed on cells of the megakaryocytic lineage, but its presence on hematopoietic progenitors has been a controversial issue. To resolve this ambiguity unequivocally, we performed clonogenic assays and intrathymic cell-transfer experiments in congenic animals. As the ontogeny of the avian hematopoietic system is well documented, we used this experimental model to trace GPIIb-IIIa expression during embryogenesis. Consequently, we now report that the GPIIb-IIIa integrin is expressed as early as embryonic day 3.5 (E3.5) to 4 in intraaortic hematopoietic clusters, the first site of intraembryonic hematopoietic progenitor emergence, and later in E6 paraaortic foci. Myeloid and erythroid progenitors were also detected within the GPIIb-IIIa+ CD45+ population isolated from the E3.5 to 4 aortic area, while in embryonic and adult bone marrow, myeloid, erythroid, and T-cell progenitors were present in the GPIIb-IIIa+ c-kit+ population. Furthermore, we also provide the first evidence, that GPIIb-IIIa+ bone marrow cells can differentiate into T cells. Hence, GPIIb-IIIa can be used as a marker for multilineage hematopoietic progenitors, permitting identification of early intraembryonic sites of hematopoiesis, as well as the isolation of embryonic and adult hematopoietic progenitors.
Hematopoietic Growth Factors Signal Through the Formation of Reactive Oxygen Species
Authors: Sattler Martin , Winkler Thomas , Verma Shalini , Byrne Christopher H. , Shrikhande Gautam , Salgia Ravi , Griffin James D. 
Published: October 2019

AbstractHematopoietic growth factors (HGFs) stimulate growth, differentiation, and prevent apoptosis of progenitor cells. Each growth factor has a specific cell surface receptor, which activates both unique and shared signal transduction pathways. We found that several HGFs, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), steel factor (SF), and thrombopoietin (TPO) induce a rapid increase in reactive oxygen species (ROS) in quiescent cells. In an effort to understand the potential biochemical and biological consequences of increased ROS in these cells, we exposed growth factor-deprived cells to hydrogen peroxide (H2O2) at concentrations that increased intracellular ROS. H2O2 induced a dose-dependent increase in tyrosine phosphorylation, including increased tyrosine phosphorylation of the GM-CSF receptor beta chain (βc), STAT5, and other signaling proteins. H2O2 also induced expression of the early response gene c-FOS, and G1- to S-phase transition, but not S- to G2/M-phase transition of MO7e cells. The cell permeable antioxidant pyrrolidine dithiocarbamate (PDTC) decreased the intracellular levels of ROS and inhibited tyrosine phosphorylation induced by GM-CSF in MO7e cells, suggesting that ROS generation plays an important role in GM-CSF signaling. Consistent with this notion, PDTC and two other antioxidants, N-acetyl cysteine and 2-mercaptoethanol, reduced growth and viability of MO7e cells. These results suggest that generation of ROS in response to HGFs may contribute to downstream signaling events, especially those involving tyrosine phosphorylation.
Promoter Element for Transcription of Unrearranged T-Cell Receptor β-Chain Gene in Pro-T Cells
Authors: Doty Raymond T. , Xia Dong , Nguyen Suzanne P. , Hathaway Tanya R. , Willerford Dennis M. 
Published: October 2019

AbstractThe hallmark of T- and B-lymphocyte development is the rearrangement of variable (V), diversity (D), and joining (J) segments of T-cell receptor (TCR) and immunoglobulin (Ig) genes to generate a diverse repertoire of antigen receptor specificities in the immune system. The process of V(D)J recombination is shared in the rearrangement of all seven antigen receptor genes and is controlled by changes in chromatin structure, which regulate accessibility to the recombinase apparatus in a lineage- and stage-specific manner. These chromatin changes are linked to transcription of the locus in its unrearranged (germline) configuration. To understand how germline transcription of the TCRβ-chain gene is regulated, we determined the structure of germline transcripts initiating near the Dβ1 segment and identified a promoter within this region. The Dβ1 promoter is active in the presence of the TCRβ enhancer (Eβ), and in this context, exhibits preferential activity in pro-T versus mature T-cell lines, as well as T- versus B-lineage specificity. These studies provide insight into the developmental regulation of TCRβ germline transcription, one of the earliest steps in T-cell differentiation.
A New Fusion Gene TPM3-ALK in Anaplastic Large Cell Lymphoma Created by a (1;2)(q25;p23) Translocation
Authors: Lamant Laurence , Dastugue Nicole , Pulford Karen , Delsol Georges , Mariamé Bernard 
Published: October 2019

AbstractAnaplastic large cell lymphomas (ALCL) are frequently associated with the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene at 5q35, which encodes a nucleolar protein involved in shuttling ribonucleoproteins from the cytoplasm to the nucleus, to the anaplastic lymphoma kinase (ALK) gene at 2p23, encoding a tyrosine kinase receptor. In this report, we describe a typical case of ALCL whose malignant cells exhibited a novel (1;2)(q25;p23) translocation. These cells expressed ALK protein, but, in contrast to t(2;5)-positive ALCL (which show cytoplasmic, nuclear, and nucleolar staining), labeling was restricted to the malignant cell cytoplasm. Using a polymerase chain reaction (PCR)-based technique to walk on chromosome 2 from the known ALK gene across the breakpoint, we showed that the gene involved at 1q25 is TPM3, encoding a nonmuscular tropomyosin. We subsequently identified, using reverse transcription-PCR analysis of cases showing similar ALK cytoplasm-restricted staining, fusion of the ALK andTPM3 genes in 2 other cases of ALCL. The TPM3 gene has been previously found in papillary thyroid carcinomas as a fusion partner with the TRK kinase gene. We showed that TPM3 is constitutively expressed in lymphoid cell lines, suggesting that, in these t(1;2)-bearing ALCL cases, the TPM3 gene contributes an active promoter for ALK expression. Activation of the ALK catalytic domain probably results from homodimerization of the hybrid protein TPM3-ALK, through the TPM3 protein-protein interaction domain. The present cases of ALCL associated with a novel t(1;2)(q25;p23) demonstrate that at least one fusion partner other than NPM can activate the intracytoplasmic domain of the ALK kinase.
LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-α
Authors: Xaus Jordi , Comalada Mònica , Valledor Annabel F. , Lloberas Jorge , López-Soriano Francisco , Argilés Josep M. , Bogdan Christian , Celada Antonio 
Published: October 2019

AbstractThe deleterious effects of lipopolysaccharide (LPS) during endotoxic shock are associated with the secretion of tumor necrosis factor (TNF) and the production of nitric oxide (NO), both predominantly released by tissue macrophages. We analyzed the mechanism by which LPS induces apoptosis in bone marrow-derived macrophages (BMDM). LPS-induced apoptosis reached a plateau at about 6 hours of stimulation, whereas the production of NO by the inducible NO-synthase (iNOS) required between 12 and 24 hours. Furthermore, LPS-induced early apoptosis was only moderately reduced in the presence of an inhibitor of iNOS or when using macrophages from iNOS -/-mice. In contrast, early apoptosis was paralleled by the rapid secretion of TNF and was almost absent in macrophages from mice deficient for one (p55) or both (p55 and p75) TNF-receptors. During the late phase of apoptosis (12-24 hours) NO significantly contributed to the death of macrophages even in the absence of TNF-receptor signaling. NO-mediated cell death, but not apoptosis induced by TNF, correlated with the induction of p53 and Bax genes. Thus, LPS-induced apoptosis results from 2 independent mechanisms: first and predominantly, through the autocrine secretion of TNF- (early apoptotic events), and second, through the production of NO (late phase of apoptosis).
Lineage-specific regulation of the murine RAG-2 promoter: GATA-3 in T cells and Pax-5 in B cells
Authors: Kishi Hiroyuki , Wei Xing-Cheng , Jin Zhe-Xiong , Fujishiro Yoshiyuki , Nagata Takuya , Matsuda Tadashi , Muraguchi Atsushi 
Published: October 2019

AbstractRecombination activating gene-1 (RAG-1) andRAG-2 are expressed in lymphoid cells undergoing the antigen receptor gene rearrangement. A study of the regulation of the mouse RAG-2 promoter showed that the lymphocyte-specific promoter activity is conferred 80 nucleotide (nt) upstream of RAG-2. Using an electrophoretic mobility shift assay, it was shown that a B-cell–specific transcription protein, Pax-5, and a T-cell–specific transcription protein, GATA-3, bind to the −80 to −17 nt region in B cells and T cells, respectively. Mutation of the RAG-2 promoter for Pax-5– and GATA-3–binding sites results in the reduction of promoter activity in B cells and T cells. These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively.
Engagement of CD11b and CD11c β2 integrin by antibodies or soluble CD23 induces IL-1β production on primary human monocytes through mitogen-activated protein kinase–dependent pathways
Authors: Rezzonico Roger , Chicheportiche Rachel , Imbert Veronique , Dayer Jean-Michel 
Published: October 2019

Abstractβ2 integrins are involved in the recruitment of leukocytes to inflammatory sites and in cellular activation. We demonstrate that ligation of CD11b (Mac-1, CR3) or CD11c (p150, CR4) alpha chains of β2 integrins by mAbs or soluble chimeric CD23 (sCD23) on human freshly isolated monocytes rapidly stimulates high levels of interleukin-1β production. This induction takes place at the transcriptional level and is regulated by members of the mitogen-activated protein kinase (MAPK) family. Indeed, stimulation of monocytes through engagement of CD11b or CD11c results in the phosphorylation and activation of ERK1, ERK2, and p38/SAPK2 MAP kinases. U0126, a potent inhibitor of the upstream activator of ERK1/2, ie, MEK1/2, suppresses IL-1β messenger RNA (mRNA) expression in a dose-dependent fashion, showing the implication of this pathway in the transcriptional control of IL-1β production. On the other hand, inhibition of p38 by SB203580 indicates that this MAPK is involved in the control of IL-1β production at both transcriptional and translational levels. Together these data demonstrate that ligation of CD11b and CD11c β2 integrins by mAbs or sCD23 fusion proteins triggers the activation of 2 distinct MAPK signaling pathways that cooperate in controlling IL-1β synthesis at different levels.
Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis
Authors: Golay Josée , Zaffaroni Luisella , Vaccari Thomas , Lazzari Manuela , Borleri Gian-Maria , Bernasconi Sergio , Tedesco Francesco , Rambaldi Alessandro , Introna Martino 
Published: October 2019

AbstractThe chimeric anti-CD20 MAb rituximab has recently become a treatment of choice for low-grade or follicular non-Hodgkin's lymphomas (FL) with a response rate of about 50%. In this report, we have investigated the mechanism of action of rituximab on 4 FL and 1 Burkitt's lymphoma (BL) cell lines, 3 fresh FL samples and normal B cells in vitro. Rituximab efficiently blocks the proliferation of normal B cells, but not that of the lymphoma lines. We did not detect significant apoptosis of the cell lines in response to rituximab alone. All cell lines were targets of antibody-dependent cellular cytotoxicity (ADCC). On the other hand, human complement-mediated lysis was highly variable between cell lines, ranging from 100% lysis to complete resistance. Investigation of the role of the complement inhibitors CD35, CD46, CD55, and CD59 showed that CD55, and to a lesser extent CD59, are important regulators of complement-mediated cytotoxicity (CDC) in FL cell lines as well as in fresh cases of FL: Blocking CD55 and/or CD59 function with specific antibodies significantly increased CDC in FL cells. We conclude that CDC and ADCC are major mechanisms of action of rituximab on B-cell lymphomas and that a heterogeneous susceptibility of different lymphoma cells to complement may be at least in part responsible for the heterogeneity of the response of different patients to rituximab in vivo. Furthermore, we suggest that the relative levels of CD55 and CD59 may become useful markers to predict the clinical response.
Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor
Authors: Stefano George B. , Cadet Patrick , Breton Christophe , Goumon Yannick , Prevot Vincent , Dessaint Jean Paul , Beauvillain Jean-Claude , Roumier Ann S. , Welters Ingeborg , Salzet Michel 
Published: October 2019

AbstractWe tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17β-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17β-estradiol conjugated to bovine serum albumin (E2-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17β-estradiol and E2-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17β-estradiol–stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca2+]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N′,N′-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca2+]i chelator, reduced [Ca2+]i in response to E2-BSA, and depleting [Ca2+]i stores abolished the effect of 17β-estradiol on NO release. Confocal photomicrographs using E2-BSA–FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase–polymerase chain reaction, human neutrophil granulocytes expressed ER but not ERβ, suggesting that ER may be the membrane receptor for 17β-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca2+]i.
Constitutive activation of the MAPK pathway mediates v-fes–induced mitogenesis in murine macrophages
Authors: Rovida Elisabetta , Marra Fabio , Baccarini Manuela , Sbarba Persio Dello 
Published: October 2019

Abstract
Fes is a nonreceptor tyrosine kinase expressed at the highest level in macrophages. We previously showed that the overexpression of c-fes in murine macrophages of the BAC-1.2F5 cell line renders these cells independent of macrophage colony-stimulating factor (MCSF) for survival and proliferation, although no direct relationship could be established between tyrosine-phosphorylated substrates of Fes- and MCSF receptor–dependent signaling and mitogenesis. In this study, we investigated whether the mitogen-activated protein kinase (MAPK) pathway is involved in the growth factor–independent growth of v-fes–overexpressing macrophages. We found a constitutively increased phosphorylation of extracellularly regulated kinase (ERK) in v-fes–overexpressing macrophages as compared with mock-infected cells. This finding was associated with activation of mitogen/extracellular signal–regulated kinase (MEK) and with constitutive localization of ERK in the nucleus. Treatment of v-fes–overexpressing cells with the MEK-specific inhibitor PD98059 markedly reduced cell growth, hyperphosphorylation, and nuclear localization of ERK, indicating that the MAPK pathway mediates the mitogenic effect of v-fes.
Posttranscriptional cell cycle–dependent regulation of human FANCC expression
Authors: Heinrich Michael C. , Silvey Kirsten V. , Stone Stacie , Zigler Amy J. , Griffith Diana J. , Montalto Michelle , Chai Lin , Zhi Yu , Hoatlin Maureen E. 
Published: October 2019

AbstractThe Fanconi Anemia (FA) Group C complementation group gene (FANCC) encodes a protein, FANCC, with a predicted Mr of 63000 daltons. FANCC is found in both the cytoplasmic and the nuclear compartments and interacts with certain other FA complementation group proteins as well as with non-FA proteins. Despite intensive investigation, the biologic roles of FANCC and of the other cloned FA gene products (FANCA and FANCG) remain unknown. As an approach to understanding FANCC function, we have studied the molecular regulation of FANCC expression. We found that although FANCCmRNA levels are constant throughout the cell cycle, FANCC is expressed in a cell cycle-dependent manner, with the lowest levels seen in cells synchronized at the G1/S boundary and the highest levels in the M-phase. Cell cycle–dependent regulation occurred despite deletion of the 5′ and 3′ FANCC untranslated regions, indicating that information in the FANCC coding sequence is sufficient to mediate cell cycle–dependent regulation. Moreover, inhibitors of proteasome function blocked the observed regulation. We conclude that FANCC expression is controlled by posttranscriptional mechanisms that are proteasome dependent. Recent work has demonstrated that the functional activity of FA proteins requires the physical interaction of at least FANCA, FANCC, and FANCG, and possibly of other FA and non-FA proteins. Our observation of dynamic control of FANCC expression by the proteasome has important implications for understanding the molecular regulation of the multiprotein complex.
Mild spherocytic hereditary elliptocytosis and altered levels of α- and γ-adducins in β-adducin-deficient mice
Authors: Muro Andrés F. , Marro Martı́n L. , Gajović Srećko , Porro Fabiola , Luzzatto Lucio , Baralle Francisco E. 
Published: October 2019

AbstractThe membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of β-adducin produces an 80% decrease of -adducin and a fourfold up-regulation of γ-adducin in erythrocytes. β-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of −/− erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of −/− mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably γ adducin) could be functional and might account for the mild phenotype.