bluuebunny/crossref_metadata_2025_split huggingface dataset:
Low levels of erythroid and myeloid progenitors in thrombopoietin-and c- mpl-deficient mice
Authors: Carver-Moore K , Broxmeyer HE , Luoh SM , Cooper S , Peng J , Burstein SA , Moore MW , de Sauvage FJ 
Published: October 2019

Abstract
Thrombopoietin (TPO), the ligand for the c-mpl receptor, has been shown to be the major regulator of platelet production. Mice deficient in either c-mpl or TPO generated by homologous recombination show a dramatic decrease in platelet counts, but other blood cell counts are normal. Because TPO treatment of myelosuppressed mice not only enhances the recovery of platelets but also accelerates erythroid recovery, we investigated the levels of myeloid and erythroid progenitor cells in TPO-or c-mpl-deficient mice. Our results show that the number of megakaryocyte, granulocyte-macrophage, erythroid, and multilineage progenitors are significantly reduced in the bone marrow, spleen, and peripheral blood of either TPO-or c-mpl-deficient mice. Administration of recombinant murine TPO to TPO-deficient mice and control littermate mice significantly increased the absolute number of myeloid, erythroid, and mixed progenitors in bone marrow and spleen. This increase was especially apparent in TPO-deficient mice where numbers were increased to a level greater than in diluent-treated control mice and approached or equaled that in the TPO-treated control mice. Moreover, TPO- administration greatly increased the number of circulating progenitors as well as platelets in both TPO-deficient and control mice. Furthermore, the megakaryocytopoietic activity of other cytokines in the absence of a functional TPO or c-mpl gene was shown both in vitro and in vivo.
Mechanism of transcriptional activation of the immediate early gene Egr- 1 in response to PIXY321
Authors: Mignacca RC , Lee HJ , Kwon EM , Sakamoto KM 
Published: October 2019

Abstract
Studies with the granulocyte-macrophage colony-stimulating factor (GM- CSF)/interleukin-3 (IL-3) fusion protein, PIXY321, demonstrated enhanced biological activity of this molecule in comparison with GM-CSF or IL-3 alone or in combination. Experiments were performed to study the mechanisms resulting in PIXY321-induced egr-1 expression in human myeloid leukemic cells (TF-1). Transfections of egr-1 promoter constructs revealed that PIXY321 stimulation resulted in fourfold induction of the -116 and -600 nucleotide (nt) constructs. We transfected a -116 nt construct containing a deletion of the cyclic AMP response element (CRE) or mutation in the serum response element (SRE) and demonstrated that both the SRE and CRE are necessary for maximal induction. However, PIXY321 stimulation resulted in 2.5-fold induction of a SRE-CRE-containing construct (P < .05), suggesting that the SRE and CRE are sufficient for PIXY321 responsiveness. Electrophoretic mobility shift assays (EMSA) revealed that the CRE binding protein (CREB) was phosphorylated on serine 133 in PIXY321-stimulated but not - unstimulated extracts from cells cultured in GM-CSF. By Western analysis and EMSA, CREB was constitutively phosphorylated in TF-1 cells grown on PIXY321 before growth factor and serum starvation. However, in TF-1 cells grown on GM-CSF before starvation, CREB phosphorylation was observed 10 minutes after PIXY321 stimulation. Further-more, ENSAs with PIXY321-stimulated and -unstimulated extracts demonstrated the presence of specific proteins that recognize the SRE. Our data demonstrate that transcriptional regulation of egr-1 by PIXY321 is mediated by the CRE and SRE.
Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6
Authors: Nurden P , Humbert M , Piotrowicz RS , Bihour C , Poujol C , Nurden AT , Kunicki TJ 
Published: October 2019

Abstract
The sequence beta (3)203–228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211–221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding- site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin- stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin- induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.
Modulated expression of notch1 during thymocyte development
Authors: Hasserjian RP , Aster JC , Davi F , Weinberg DS , Sklar J 
Published: October 2019

Abstract
The Notch gene family encodes transmembrane proteins that have been implicated in control of diverse cellular differentiation events in the fly, frog, and mouse. Mammalian Notch1 is expressed at high levels in thymus and is mutated in a subset of human T-cell acute lymphoblastic neoplasms, suggesting a role in T-cell differentiation. To investigate the patterns of expression of NOTCH1 protein in thymocytes of the developing and mature thymus, antibodies raised against NOTCH1 were used to perform immunohistochemical and flow cytometric analyses. Strong staining for NOTCH1 within the fetal murine thymus was observed as early as 13.5 days postcoitum. By 17.5 days postcoitum, preferential staining of superficial cortical thymocytes was observed, with weak staining of developing medulla. Flow cytometric analysis and immunohistochemical staining of flow-sorted cells confirmed that the highest levels of NOTCH1 expression in adult murine thymus were present in immature cortical thymocytes (CD24high, CD4-CD8-). In contrast, NOTCH1 expression was low or absent in more mature cortical thymocytes (CD24low, CD4+CD8+), whereas intermediate levels of expression were observed in CD4+CD8- and CD4-CD8+ cells. These data indicate a dynamic pattern of NOTCH1 expression during T-cell differentiation and suggest that downregulation of NOTCH1 may be required for maturation of cortical thymocytes.
PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl
Authors: Jain SK , Susa M , Keeler ML , Carlesso N , Druker B , Varticovski L 
Published: October 2019

Abstract
BCR/abl is a chimeric oncogene implicated in the pathogenesis of human chronic myelogenous leukemia. Expression of the BCR/abl gene induces hematologic malignancies in transgenic mice and transformation of interleukin-3-dependent hematopoietic cells. The mechanism of BCR/abl- mediated transformation of hematopoietic cells is poorly understood and involves activation of at least two signaling pathways, p21ras and PI 3- kinase. Here we report that PI 3,4-P2 and PI 3,4,5-P3, the enzymatic products of PI 3-kinase, accumulate in metabolically labeled transformed hematopoietic cells, in contrast to our previous report on the lack of accumulation of PI 3-kinase products in nontransformed NIH 3T3 fibroblasts that express p210 BCR/abl. Transformed cells also have increased PI 3-kinase activity in total cell extracts and membrane fractions. Activation of PI 3-kinase occurs by occupancy of SH2 domains of PI 3-kinase regulatory subunit, p85, by phosphorylated YXXM motifs. Therefore, we investigated whether BCR/abl binds to p85 and whether this binding is mediated by interaction of p85 SH2 domains with YXXM motif of BCR/abl. Association of p210 BCR/abl with p85 in immune complexes and with p85 SH2 domains was evident in hematopoietic cells that express the wt p210 BCR/abl. However, the binding of BCR/abl to p85 SH2 domains was abolished in cells expressing mutant, temperature- sensitive (ts) p210 BCR/abl in which the tyrosine in the YXXM motif of p210 BCR/abl was replaced by histidine. Despite lack of direct interaction with p85 SH2 domains, expression of ts p210 BCR/abl resulted in rapid, time-dependent activation of total and membrane- associated PI 3-kinase and increased PI 3-kinase activity in anti-P-tyr and anti-abl immunoprecipitates. These data suggest that BCR/abl- induced activation of PI 3-kinase in hematopoietic cells does not require binding of p85 SH2 domains to BCR/abl gene product and involves interaction with other tyrosine phosphorylated intermediate proteins.
Molecular basis of protein S deficiency in three families also showing independent inheritance of factor V leiden
Authors: Beauchamp NJ , Daly ME , Cooper PC , Makris M , Preston FE , Peake IR 
Published: October 2019

Abstract
The molecular basis of type I or III Protein S deficiency has been investigated in three kindred also showing independent inheritance of factor V (FV) Leiden. A T to C transition in codon 570 (Met-->Thr) was identified in the propositi and shown to segregate with protein S deficiency in all but one of the affected members of two kindred. This individual was heterozygous for a second transition (C to T) causing substitution of serine 624 by leucine. A second member of the same family, with markedly reduced free protein S levels when compared with affected relatives, was heterozygous for both mutations. Haplotype analysis of individuals with the mutated ATG570ACG allele in the two kindred suggested they may have been related by a common ancestor. A G to A transition resulting in substitution of cysteine 145 by tyrosine was detected in the third kindred. All mutations are believed to interfere with protein S binding to C4b-binding protein resulting in reduced free protein S levels. Of the five individuals studied who had experienced thrombotic events, three had combined protein S deficiency and FV Leiden reemphasising the importance of FV Leiden as an additional risk factor for thrombosis in protein S deficiency.
Deficient expression of adhesion molecules by human CD5- B lymphocytes both after bone marrow transplantation and during normal ontogeny
Authors: Parra C , Roldan E , Brieva JA 
Published: October 2019

Abstract
Despite the relatively early reconstitution of blood B-lymphocyte counts observed in patients treated with bone marrow transplantation (BMT), these patients undergo a prolonged phase of humoral immunodeficiency. Adhesion molecules perform relevant functions in many cell types. The present study examines the expression of several adhesion molecules on human B lymphocytes newly formed after BMT. Blood B cells from 38 patients were studied by flow cytometry and three-color analysis. Blood CD5- B lymphocytes obtained at an early stage after BMT (2 to 4 months) showed a markedly low expression of the adhesion molecules CD54, CD44, CD11a, and CD62L. However, these cells exhibited a normal expression of other molecules including CD29, CD19, CD20, and DR. This deficiency was progressively corrected, reaching normal levels in the late post-BMT period (12 to 15 months). In contrast, CD54, CD44, CD11a, and CD62L expression on the patients' CD5+ B lymphocytes was found to be consistently normal. Deficient adhesion molecule expression on CD5- B cells in the early post-BMT period was similarly observed in patients treated with either an allo-BMT (n = 24) or an auto-BMT (n = 14). Because the post-BMT period mimics normal ontogeny, adhesion molecule expression was also investigated in cord-blood B lymphocytes. Cord-blood CD5- B lymphocytes, in contrast to CD5+, also expressed CD54, CD44, CD11a, and CD62L at levels much lower than those found in normal adults. Present data suggest that progressive expression of CD54, CD44, CD11a, and CD62L seems to be a part of the maturational program of CD5- B lymphocytes during both post-BMT and normal development periods. This observation may help to explain the humoral immunodeficiency observed in both conditions.
Activation of the CD95 (APO-1/Fas) system in T cells from human immunodeficiency virus type-1-infected children
Authors: Baumler CB , Bohler T , Herr I , Benner A , Krammer PH , Debatin KM 
Published: October 2019

Abstract
Increased apoptosis of CD4+ T cells is considered to be involved in CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1)- infected individuals progressing toward acquired immunodeficiency syndrome (AIDS). We have recently shown that CD95 (APO-1/Fas) expression is strongly increased in T cells of HIV-1-infected children. In this report we provide further evidence for a deregulated CD95 system in AIDS. CD95 expression in HIV-1+ children is not restricted to previously activated CD45RO+ T cells but is also increased on freshly isolated naive CD45RA+ T cells. In addition, specific CD95-mediated apoptosis is enhanced in both CD4+ and CD8+ T cells. Furthermore, levels of CD95 ligand mRNA are profoundly increased. Specific T-cell receptor/CD3-triggered apoptosis in HIV-1+ children is more enhanced in CD8+ than in CD4+ T cells. Accelerated activation induced cell death of T cells could partially be inhibited by blocking anti-CD95 antibody fragments. These data suggest an involvement of the CD95 receptor/ligand system in T-cell depletion and apoptosis in AIDS and may open new avenues of rational intervention strategies.
Lack of the t(2;5) or other mutations resulting in expression of anaplastic lymphoma kinase catalytic domain in CD30+ primary cutaneous lymphoproliferative disorders and Hodgkin's disease
Authors: Wood GS , Hardman DL , Boni R , Dummer R , Kim YH , Smoller BR , Takeshita M , Kikuchi M , Burg G 
Published: October 2019

Abstract
The t(2;5) (p23;q35) chromosomal translocation has been found in a high proportion of lymph node-based CD30+ large cell lymphomas of T-cell lineage. This translocation is believed to result in the expression of a fusion protein containing the catalytic domain of anaplastic lymphoma kinase (ALK) under the control of the promoter for nucleophosmin, a nucleolar phosphoprotein. Expression of ALK activity, which does not normally occur in lymphocytes, is postulated to be involved in the pathogenesis of lymphomas bearing the t(2;5) translocation. Several primary cutaneous lymphoproliferative disorders and Hodgkin's disease are also known to contain CD30+ large lymphoid cells. To determine the role of the t(2;5) translocation in these diseases, we developed a DNA- based polymerase chain reaction (PCR)/Southern blot assay to detect this translocation at the genomic level in lymphomatoid papulosis (14 cases), primary cutaneous CD30+ large cell lymphoma of T-lineage (10 cases) and Hodgkin's disease (13 cases). Two cases of pityriasis lichenoides were also studied. The t(2;5) translocation was not present in any of these specimens. To determine if some other somatic mutation might have resulted in inappropriate expression of ALK catalytic domain, we devised an RNA-based reverse transcriptase-PCR assay to detect transcripts encoded by this ALK region. None were found in the six additional cases of lymphomatoid papulosis that were studied. In aggregate, these results strongly suggest that inappropriate expression of ALK is not involved in the pathogenesis of these CD30+ lymphoproliferative disorders, and that lymph node-based CD30+ large cell lymphoma is a disease that is biologically distinct from skin- based CD30+ lymphoproliferative disorders and Hodgkin's disease. Using methods developed for this report, we also cloned and sequenced the t(2;5) genomic junctional sequences present in the SUP-M2 and SU-DHL-1 cell lines. These intron sequences will be useful for mapping t(2;5) breakpoint clusters.
Molecular Pathogenesis of Type I Congenital Plasminogen Deficiency: Expression of Recombinant Human Mutant Plasminogens in Mammalian Cells
Authors: Azuma Hiroyuki , Mima Nobuaki , Shirakawa Mitsuo , Miyamoto Kazumasa , Yamaguchi Hiroshi , Mitsui Takao , Shigekiyo Toshio , Saito Shiro 
Published: October 2019

AbstractWe previously reported the genetic abnormality in a Japanese family with type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation. To characterize the molecular pathogenesis of the disease in this family, we expressed recombinant human wild-type and mutant (rS572P) plasminogens in COS-1 cells. Activation-resistant wild-type and mutant plasminogen stable transfectants in CHO-K1 cells also were established. Transient transfection and metabolic labeling experiments followed by immunoprecipitation analysis showed that the mutant plasminogen was secreted from COS-1 cells in reduced amounts, compared with the wild type. Endo H digestion of the wild-type and mutant plasminogen showed no shift in their migrations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, indicating that both contain complex type oligosaccharide structures and could therefore be secreted. Furthermore, the secretion of activation-resistant mutant plasminogen was significantly reduced. Pulse-chase experiments and Northern blot analysis showed that the impaired secretion of the mutant plasminogen was the consequence of the accumulation of the mutant protein inside the cells but not of reduced plasminogen mRNA. Immunocytochemical staining of stable transfectants also revealed that CHO-K1 cells expressing the activation-resistant mutant plasminogen stained mainly in the perinuclear area, suggesting delayed processing of the mutant protein in the intracellular transport pathway. We conclude that the impaired secretion of mutant plasminogen, due to intracellular accumulation, is the molecular pathogenesis of type I congenital plasminogen deficiency caused by a Ser572 to Pro572 mutation.
Transfer of the Interleukin-2 Gene Into Human Cancer Cells Induces Specific Antitumor Recognition and Restores the Expression of CD3/T-Cell Receptor Associated Signal Transduction Molecules
Authors: Guarini Anna , Riera Ludovica , Cignetti Alessandro , Montacchini Laura , Massaia Massimo , Foa Robert 
Published: October 2019

AbstractNormal peripheral blood mononuclear cells (PBMC) were cocultured with a human lung cancer cell line (LC89) transduced with the interleukin-2 (IL-2), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF ), and tumor necrosis factor-α (TNF-α) genes to evaluate the capacity of the engineered cells to: allow survival of CD3+ and CD56+ cells, generate cytotoxic effectors with HLA class I restricted and unrestricted antitumor activity, and interfere in the molecular organization of the CD3/T-cell receptor associated signal transduction machinery. When PBMC were cultured up to 3 weeks with IL-2 releasing LC89 cells (LC89/IL-2), the number of viable CD3+ and CD56+ lymphocytes was much greater than in cultures with parental cells or with LC89 cells transduced with the other cytokine genes. After 1 week of coculture, a variable degree of restricted and unrestricted killing directed against different targets was observed. When the cultures were prolonged up to 3 weeks, LC89/IL-2 cells induced a marked increase in specific cytotoxic activity, which was coupled to a further enhancement of unrestricted lytic function. In the presence of LC89/IL-7 cells the degree of specific lysis remained unchanged, whereas unrestricted effectors were markedly decreased. No cytotoxic activity could be induced by LC89/GM-CSF and LC89/TNF-α cells in the few lymphocytes surviving after 3 weeks of culture. Coculture of parental LC89 cells with PBMC was consistently associated with a downmodulation in the expression of the CD3 ζ chain, as well as of the tyrosine kinases p56lck and ZAP-70. On the contrary, LC89/IL-2 cells, and not LC89 cells transduced with the IL-7, GM-CSF, or TNF-α gene, were capable of reverting the immunosuppressive effect exerted by the tumor cells. This protective effect could be maintained in cultures prolonged up to 4 weeks. When the same cultures were set up in Transwell, ie, with a membrane separation between cancer cells and PBMC, the expression of the CD3 ζ chain and of the p56lck and ZAP-70 tyrosine kinases remained unchanged under all culture conditions, indicating that the downmodulation of T-cell signal transduction molecules requires a direct cell to cell contact. These results show that transfer of the IL-2 gene into the DNA of human cancer cells promotes both restricted and unrestricted antitumor activity, and is capable of restoring and maintaining the expression of molecules involved in the process of T-cell mediated tumor cell recognition, thus underlining the potential role of the IL-2 gene in the design of vaccination protocols with cytokine gene transduced cancer cells.
Interleukin-6 Inhibits Fas-Induced Apoptosis and Stress-Activated Protein Kinase Activation in Multiple Myeloma Cells
Authors: Chauhan Dharminder , Kharbanda Surender , Ogata Atsushi , Urashima Mitsuyoshi , Teoh Gerrard , Robertson Michael , Kufe Donald W. , Anderson Kenneth C. 
Published: October 2019

AbstractFas belongs to the family of type-1 membrane proteins that transduce apoptotic signals. In the present studies, we characterized signaling during Fas-induced apoptosis in RPMI-8226 and IM-9 multiple myeloma (MM) derived cell lines as well as patient plasma cell leukemia cells. Treatment with anti-Fas (7C11) monoclonal antibody (MoAb) induced apoptosis, evidenced by internucleosomal DNA fragmentation and propidium iodide staining, and was associated with increased expression of c-jun early response gene. We also show that anti-Fas MoAb treatment is associated with activation of stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK); however, no detectable increase in extracellular signal-regulated kinases (ERK1 and ERK2) activity was observed. Because interleukin-6 (IL-6) is a growth factor for MM cells and inhibits apoptosis induced by dexamethasone and serum starvation, we examined whether IL-6 affects anti-Fas MoAb-induced apoptosis and activation of SAPK or p38 MAPK in MM cells. Culture of MM cells with IL-6 before treatment with anti-Fas MoAb significantly reduced both DNA fragmentation and activation of SAPK, without altering induction of p38 MAPK activity. These results therefore suggest that anti-Fas MoAb-induced apoptosis in MM cells is associated with activation of SAPK, and that IL-6 may both inhibit apoptosis and modulate SAPK activity.