bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nitric oxide interactions with cobalamins: biochemical and functional consequences
Authors: Brouwer M , Chamulitrat W , Ferruzzi G , Sauls DL , Weinberg JB 
Published: October 2019

Abstract
Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.
Prevention of Graft-Versus-Host Disease in Mice Using a Suicide Gene Expressed in T Lymphocytes
Authors: Cohen José L. , Boyer Olivier , Salomon Benoı̂t , Onclercq Rosine , Charlotte Frédéric , Bruel Sylvie , Boisserie Gilbert , Klatzmann David 
Published: October 2019

AbstractAlloreactive T cells present in a bone marrow transplant are responsible for graft-versus-host disease (GVHD), but their depletion is associated with impaired engraftment, immunosuppression, and loss of the graft-versus-leukemia effect. We developed a therapeutic strategy against GVHD based on the selective destruction of these alloreactive T cells, while preserving a competent T-cell pool of donor origin. We generated transgenic mice expressing in their T lymphocytes the Herpes simplex type 1 thymidine kinase (TK) suicide gene that allows the destruction of dividing T cells by a ganciclovir treatment. T cells expressing the TK transgene were used to generate GVHD in irradiated bone marrow grafted mice. We show that a short 7-day ganciclovir treatment, initiated at the time of bone marrow transplantation, efficiently prevented GVHD in mice receiving TK-expressing T cells. These mice were healthy and had a normal survival. They maintained a T-cell pool of donor origin that responded normally to in vitro stimulation with mitogens or third party alloantigens, but were tolerant to recipient alloantigens. Our experimental system provides the proof of concept for a therapeutic strategy of GVHD prevention using genetically engineered T cells.
Vascular Cell Adhesion Molecule-1 Is Expressed by Cortical Thymic Epithelial Cells and Mediates Thymocyte Adhesion. Implications for the Function of α4β1 (VLA4) Integrin in T-Cell Development
Authors: Salomon Daniel R. , Crisa Laura , Mojcik Christopher F. , Ishii Jennifer K. , Klier George , Shevach Ethan M. 
Published: October 2019

AbstractT-cell development requires a series of discrete selection and activation signals delivered to maturing progenitors in the thymic cortex and medulla. We have previously shown the constitutive activity of the integrin, α4β1 (VLA4), on a unique subpopulation of immature cortical thymocytes and proposed a role for integrin-mediated adhesion in positive selection by cortical epithelium. In the present report we show that thymic epithelial cell lines express vascular cell adhesion molecule-1 (VCAM-1) a high-affinity ligand for α4β1, and that VCAM-1 mediates thymocyte binding to these lines. Immunohistochemistry and confocal microscopy show that VCAM-1 is selectively expressed in situ by thymic epithelium in the cortex and corticomedullary junction, two locations at which VCAM-1 could determine the interaction between immature thymocytes and selecting elements on epithelial cells. In parallel, we confirmed that fibronectin (FN), the alternative ligand for α4β1, is expressed predominantly in the medulla. These results suggest that VCAM-1 is an adhesive ligand in the thymic cortex for the activated form of α4β1 constitutively expressed during development by immature double positive thymocytes. The structural segregation of the alternative ligand, FN, to the medulla suggests that medullary FN may regulate the migration, development, and export of more mature thymocytes.
Splenectomy in Far-Advanced Hodgkin’s Disease
Authors: SYKES MARGUERITE P. , KARNOFSKY DAVID A. , MCNEER GORDON P. , CRAVER LLOYD F. 
Published: October 2019

Abstract
Five patients with far-advanced Hodgkin’s disease and with evidence of hematopoietic failure were treated by splenectomy in order to determine whether: (1) the course of the disease could be modified, (2) the hematologic picture improved, and (3) responsiveness to nitrogen mustard or x-ray therapy restored. These patients showed a transient slight improvement in their hematologic status, but the course of the disease possibly was accelerated, and the patients all died within thirteen weeks, without showing renewed suitability or increased responsiveness to therapy.
On the basis of our data and a review of thirty cases from the literature, it is concluded that splenectomy is not a useful procedure its Hodgkin’s disease, except for certain specific indications. These may be: (1) an apparently solitary splenic tumor; (2) acquired hemolytic anemia, although this process may be better controlled in some cases by treating the underlying Hodgkin’s disease with x-rays, nitrogen mustard, or triethylene melamine ; (3) thrombocytopenic purpura, which appears to be more profound than is to be expected from the severity and extent of Hodgkin’s disease; and (4) hypersplenism. Hematopoietic depression in the vast majority of patients with Hodgkin’s disease, however, cannot be attributed to splenic overactivity or malfunction.
Stem Cell Factor Influences Mast Cell Mediator Release in Response to Eosinophil-Derived Granule Major Basic Protein
Authors: Furuta Glenn T. , Ackerman Steven J. , Lu Lei , Williams Rachel E. , Wershil Barry K. 
Published: October 2019

Stem cell factor (SCF) is an important mast cell growth, differentiation, and survival factor. We investigated whether SCF influenced the response of mouse mast cells to an IgE-independent stimulus, eosinophil-derived granule major basic protein (MBP). Mouse bone marrow cultured mast cells (BMCMC) were derived in either concanavalin-stimulated mouse spleen conditioned medium (CM) or SCF. The cloned growth, factor-independent mast cell line Cl.MC/C57.1 was also studied. BMCMC in SCF exhibited cytochemical staining properties, protease and histamine content, and increased serotonin uptake consistent with more mature differentiated mast cells as compared with BMCMC in CM or Cl.MC/ C57.1 cells. BMCMC in SCF released serotonin,14C-labeled arachidonic acid metabolites and tumor necrosis factor-α (TNF-α) on stimulation with MBP, while no response was seen from either BMCMC in CM or Cl.MC/C57.1 cells. All three mast cell populations released mediators on stimulation with the cationic MBP analog, poly-L-arginine, indicating that the cationic charge did not explain the selective response of BMCMC in SCF to eosinophil-derived granule MBP. These findings show that SCF significantly influences mast cell differentiation and the responsiveness of mast cells to eosinophil-derived granule MBP.© 1998 by The American Society of Hematology.
Early Detection of Relapse by Prospective Reverse Transcriptase-Polymerase Chain Reaction Analysis of the PML/RARα Fusion Gene in Patients With Acute Promyelocytic Leukemia Enrolled in the GIMEMA-AIEOP Multicenter “AIDA” Trial
Authors: Diverio Daniela , Rossi Vincenzo , Avvisati Giuseppe , DeSantis Silvia , Pistilli Alessandra , Pane Fabrizio , Saglio Giuseppe , Martinelli Giovanni , Petti Maria Concetta , Santoro Alessandra , Pelicci Pier Giuseppe , Mandelli Franco , Biondi Andrea , Coco Francesco Lo 
Published: October 2019

Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARα fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARα+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARα, as assessed by serial dilution experiments, was 10−4. All patients were in hematologic remission and tested PCR− at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR− in ≥2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARα during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up.
© 1998 by The American Society of Hematology.
Characterization of the Role of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor α Subunit in the Activation of JAK2 and STAT5
Authors: Doyle Sean E. , Gasson Judith C. 
Published: October 2019

The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) consists of an alpha (GMRα) and a common beta (βc) subunit. The intracellular domain of βc has been extensively characterized and has been shown to be critical for the activation of both the JAK/STAT and MAP kinase pathways. The function of the intracellular domain of GMRα, however, is not as well characterized. To determine the role of this domain in GMR signaling, an extensive structure-function analysis was performed. Truncation mutants α362, α371, and α375 were generated, as well as the site-directed mutants αVQVQ and αVVVV. Although α375β, αVQNQβ, and αVVVVβ stimulated proliferation in response to human GM-CSF, the truncation mutants α362β and α371β were incapable of transducing a proliferative signal. In addition, both α371 and αVVVV were expressed at markedly reduced levels, indicating the importance of residues 372 to 374 for proper protein expression. More importantly, we show that GMRα plays a direct role in the activation of the JAK/STAT pathway, and electrophoretic mobility shift assays (EMSA) indicate that both GMRα and βc play a role in determining the STAT5 DNA binding complex activated by the GMR. Thus, the intracellular domain of the human GMRα is important for activation of the JAK/STAT pathway and protein stabilization.© 1998 by The American Society of Hematology.
Use of the Microculture Kinetic Assay of Apoptosis to Determine Chemosensitivities of Leukemias
Authors: Kravtsov Vladimir D. , Greer John P. , Whitlock James A. , Koury Mark J. 
Published: October 2019

Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate–labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell lines and patient samples indicate that ANLL cells are more sensitive than ALL cells to all three of these chemotherapeutic agents. However, for individual patients the chemosensitivities varied significantly among the three chemotherapeutic agents. These varying responses to IDR, DNR, and MTA indicate that the MiCK assay results can be of potential use in designing a treatment regimen for a specific patient with acute leukemia. Among several drugs of presumed similar efficacy, the MiCK assay can permit the selection of the specific chemotherapeutic agent that causes the most apoptosis in the patient's leukemic cells.© 1998 by The American Society of Hematology.
Fas-Mediated Modulation of Bcr/Abl in Chronic Myelogenous Leukemia Results in Differential Effects on Apoptosis
Authors: Selleri Carmine , Maciejewski Jaroslaw P. , Pane Fabrizio , Luciano Luigia , Raiola Anna Maria , Mostarda Ilaria , Salvatore Francesco , Rotoli Bruno 
Published: October 2019

Fas-R is expressed constitutively in CD34+ cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that α-interferon (IFN-α) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-α can prime CML cells for the effects of Fas, the response to IFN-α in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34+ CML cells after Fas-R triggering and the clinical response to IFN-α. After priming with IFN-α, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-α; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-α. In responders to IFN-α, Fas-R agonist induced dose-dependent apoptosis of CD34+ cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-α, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and nobcr/abl downmodulation was observed. Finally, we measuredbcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/ablprotein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-α treatment.© 1998 by The American Society of Hematology.
Ex Vivo Expansion of Genetically Marked Rhesus Peripheral Blood Progenitor Cells Results in Diminished Long-Term Repopulating Ability
Authors: Tisdale J.F. , Hanazono Y. , Sellers S.E. , Agricola B.A. , Metzger M.E. , Donahue R.E. , Dunbar C.E. 
Published: October 2019

The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)–mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.This is a US government work. There are no restrictions on its use.
The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells
Authors: Harris Kevin W. , Hu Xian-Jue , Schultz Suzanne , Arcasoy Murat O. , Forget Bernard G. , Clare Nanette 
Published: October 2019

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)–dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation.© 1998 by The American Society of Hematology.