bluuebunny/crossref_metadata_2025_split huggingface dataset:
Imatinib Mesylate (STI571) Abrogates the Resistance to Doxorubicin in K562 Chronic Myeloid Leukemia Cells by Inhibition of BCR/ABL Kinase-Mediated DNA Repair.
Authors: Majsterek Ireneusz , Blasiak Janusz , Slupianek Artur , Skorski Tomasz 
Published: October 2019

Abstract
Imatinib mesylate (STI571), a specific inhibitor of the BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanisms by which inhibition of BCR/ABL activity results in pharmacological responses remain unknown. BCR/ABL-positive human CML cells resistant to doxorubicin K562doxoR and their sensitive K562doxoS counterparts were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. We examined kinetics of DNA repair after cell treatment with the drug by the alkaline comet assay. MTT assay was used to estimate resistance against doxorubicin and Western Blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562doxoR cells exhibited accelerated kinetics of DNA repair in comparison to doxorubicin-sensitive K562doxoS cells. Inhibition of BCR/ABL kinase in K562doxoR cells with 1 μM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA repair pathways, which in turn may enhance drug sensitivity of leukemia cells.
Transcriptional Programming in Quiescent Human Hematopoietic Stem Cells - Implications for Regulation of Lineage Commitment Decisions.
Authors: Pina Cristina , Enver Tariq 
Published: October 2019

Abstract
Human cord blood-derived CD133+G0 cells are a primitive population highly enriched in hematopoietic stem cells (HSC). We used this population to investigate the molecular characteristics of primitive human HSC, and, in particular, to unveil how different cell fates of quiescence, self-renewal and lineage commitment and differentiation are regulated at the molecular level. We isolated cord blood CD133+ cells in the G0 and G1 compartments of the cell cycle on the basis of low or high RNA content, respectively, as detected by Pyronin Y staining. More than 98% of the CD133+G0 cells were Ki67-negative, and at least 90% did not express CD38, thus confirming the quiescent and primitive status of the cells. Consistent with earlier findings showing that CD133+G0 cells have the highest reported frequency of LTC-IC (1), our quiescent population presented a significantly higher frequency of LTC-IC when compared to CD133+G1 cells. We further showed that CD133+G0 cells had significantly higher colony-forming capacity in progenitor assays and a higher CFU-Mix content. Initial RT-PCR analysis revealed that while both compartments express the erythroid marker beta-globin, myeloid MPO and lymphoid IL7R can only be observed in CD133+G1 cells. This suggests a hierarchy of commitment decisions in relation to cell cycle that places the erythroid signature upstream of myelo-lymphoid differentiation and may be in agreement with revised models of the hematopoietic differentiation tree recently proposed in mouse (2). This hypothesis is currently being assessed at the single-cell level. We next compared the overall gene expression programmes of CD133+G0 and G1 populations using global profiling. Consistent with the sorting criteria, CD133+G1-enriched transcripts have a comparatively higher frequency of cell cycle, protein synthesis and RNA processing, and metabolism-associated genes, which underlines the robustness of the data. The CD133+G1 population is associated with a lymphoid signature, including immunoglobulin heavy and light chains, and the SLAM family member CD48, which is consistent with the revised hierarchy of commitment decisions discussed above. Functional categories comparatively over-represented amongst CD133+G0-enriched genes include transcriptional regulation and signal transduction, suggesting that primitive quiescent HSC are ready to respond to a variety of cues that modulate alternative fate decisions. Since the transcriptional profile of a given population of cells may not reflect the transcriptional profile of each individual cell, we are currently analyzing the expression patterns of CD133+G0-enriched transcription factors (TF) at the single-cell level. This approach may help define subpopulations of cells with unique molecular signatures and suggest functional subcompartments within an otherwise heterogeneous population of primitive hematopoietic cells.
Antagonistic Effect of C/EBPα and Pax5 in Myeloid or Lymphoid Lineage Choice in Common Lymphoid Progenitors.
Authors: Fleischman Angela G. , Hsu Chia-Lin , Lai Anne Y. , Matsumoto Yukie , Weissman Irving L. , Kondo Motonari 
Published: October 2019

Abstract
Lymphoid lineage committed progenitors, such as common lymphoid progenitors (CLPs), maintain a latent myeloid differentiation potential, which can be initiated by stimulation through exogenously expressed cytokine receptors including IL-2 receptors. The transcription factor C/EBPa is promptly upregulated in CLPs upon ectopic IL-2 stimulation. We show that enforced C/EBPa expression is sufficient to initiate myeloid differentiation from CLPs, as well as from proT and proB cells, even though proB cells do not give rise to myeloid cells following ectopic IL-2 stimulation. Expression of Pax5, a B lymphoid affiliated transcription factor, is completely suppressed by enforced C/EBPa but not by ectopic IL-2 stimulation in proB cells. Introduction of Pax5 blocks ectopic IL-2 receptor-mediated myeloid lineage conversion in CLPs. These data suggest that C/EBPa is a proximal target of cytokine induced lineage conversion in lymphoid progenitors. Furthermore, complete loss of Pax5 expression triggered by upregulation of C/EBPa is a critical event for lineage conversion from lymphoid to myeloid lineage in CLPs and proB cells.
Negative Regulation of c-Kit Is Dependent on Direct Binding of Cbl to Tyrosines 568 and 936.
Authors: Masson Kristina , Heiss Elke , Ronnstrand Lars 
Published: October 2019

Abstract
C-Kit belongs to the family of type III receptor tyrosine kinases and is mainly expressed in hematopoietic precursor cells. Binding of stem cell factor (Kit ligand) leads to receptor dimerization, activation of intrinsic tyrosine kinase activity and cross-autophosphorylation of receptor tyrosine residues which provide specific docking sites for molecules that transduce the signal further downstream. Signaling through c-Kit results in several different cellular responses, most of which are directly involved in growth control and survival. Negative regulation of the receptor activity is therefore of much importance concerning pathogenic processes such as tumorigenesis in which activated c-Kit often plays a role. Here we present data showing that the ubiquitin E3 ligase Cbl must be activated by the Src family kinases and recruited to c-Kit in order to trigger receptor degradation. We demonstrate that the autophosphorylation sites Y568 and Y936 in c-Kit, previously reported to be binding sites for APS, Src or Grb2, respectively, are involved in Cbl recruitment and activation, possibly in a direct manner. I571 and L939 of c-Kit hereby determine the specificity for binding the N-terminal tyrosine kinase binding domain of Cbl. We show that activation of Cbl by Src family kinase members on one hand and direct or indirect recruitment of active Cbl to the pY568/I571 and pY936/L939 motifs of c-Kit on the other, are separable events and are both necessary for Cbl-mediated c-Kit degradation. Consistently, both the Y568/570F double mutant lacking Src kinase activity and the I571A/L939A double mutant lacking Cbl-specific interaction sites fail to ubiquitinate and degrade c-Kit upon ligand stimulation in stably transfected PAE and Ba/F3 cells. Thus, we could reveal novel aspects of the dynamic process of docking and activating signaling molecules at receptor phosphotyrosines that negatively regulate c-Kit-mediated responses. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor subsequently is targeted for lysosomal degradation. Taken together, our findings reveal novel insights on how c-Kit -mediated signal transduction is negatively regulated by Cbl.
T Cells Response and Autoantibodies to Human Heat Shock Proteins in Aplastic Anemia Patients.
Authors: Cheng Yunfeng , Tang Yong , Young Neal S. 
Published: October 2019

Abstract
Heat shock proteins (HSP) have been implicated in autoimmune diseases such as type I diabetes mellitus, systemic lupus erythematosus, and multiple sclerosis, in which T cell proliferative responses or autoantibodies towards endogenous HSP have been detected (Journal of Autoimmunity2003;20:313). HSP70 can function as an endogenous ‘danger’ signal, acting on antigen-presenting cells and critically influencing the decision between induction of tolerance and immunity upon antigen encounter (Millar et al. Nature Medicine2003; 9:1469). We studied T-cell proliferative responses and auto-antibodies to human HSP60, HSP70 and HSP90 proteins in 20 newly diagnosed aplastic anemia patients, peripheral blood was obtained (11 females, 9 males; age average 36.1±19 years). A non-isotopic immunoassay was used for BrdU incorporation into proliferative T cells and ELISA to measure HSP antibody titer. T cell proliferation was measured as the Eu-fluorescence in a time-resolved fluorometer. A positive result was defined as > 2 standard deviations (SD) from the mean of the controls. T-cell responses to HSP70 in the patient group (N=20; mean±SD Eu-fluorescence= 47,129±36,248) were significantly greater than those of the control group (N=18 healthy adult; mean Eu-fluorescence= 23,941±12,996; p=0.01). Fifty percent of the patients showed increased T cell proliferation after HSP70 stimulation compared to 5% in the control group (p=0.03). T-cell responses of the patient group to HSP90 and HSP60 were similar to those of the control group. Twenty percent of patients showed increased T cell proliferation to HSP 60 and HSP 90 stimulation compared to 5% in the control group (p=0.363). HSP antibody (IgG/A/M) seropositivity was 25% to HSP60, 50% to HSP70, and 5% to HSP90 in patients and 8% to HSP60, 0% to HSP70, and 0% to HSP90 in controls. Heightened autoimmunity to HSP70, but not to HSP60 and HSP90, is a feature of acquired aplastic anemia at presentation.
Mechanism of Selectivity of Arsenic Trioxide for Acute Promyelocytic Leukemia Cells from Screening a Genome Wide Set of Saccharomyces cerevisiae Deletion Strains.
Authors: Dilda Pierre J. , Perrone Gabriel G. , Dawes Ian W. , Hogg Philip J. 
Published: October 2019

Abstract
All-trans retinoic acid (ATRA) targets the underlying molecular lesion in acute promyelocytic leukaemia (APL) and leads to differentiation of leukemic blasts into mature granulocytes. Treatment with ATRA, however, is associated with the retinoic acid syndrome and relapse is also a problem. In relapsed patients, arsenic trioxide is considered the treatment of choice. Arsenic trioxide is a trivalent arsenical that selectively kills APL cells. To better understand how arsenic trioxide perturbs cellular functions we chose to screen a genome-wide set of Saccharomyces cerevisiae deletion strains for sensitivity or resistance to the drug. The idea was that genes whose loss conferred sensitivity to the drug would reflect mechanisms of cell death, while genes whose loss conferred resistance would suggest mechanisms of selectivity. 7.6% of the 4,564 mutants were more sensitive to arsenic trioxide than the wild-type strain, while 1.5% was more resistant. In accordance with published studies in mammalian cells, yeast mutants lacking genes required for cytoskeleton stability, response to oxidative stress and DNA repair were prominent in the sensitive list. The most prominent genes in the resistant list were that encoding the plasma membrane transporter, Fps1, Hog1 and repressors of Hog1. Fps1 is an aquaglyceroporin that mediates plasma membrane glycerol flux and uptake of inorganic arsenic in yeast. Its activity is controlled by the high osmolarity MAPK signalling or HOG pathway. There was a clear correlation between genes involved in modulating HOG pathway activity and altered arsenic trioxide tolerance in yeast. In mammalian cells, aquaglyceroporin 9 is the human homolog of yeast Fps1 and the p38 MAPK signalling cascade constitutes the analogous pathway to HOG. APL is characterized by chromosomal rearrangements of 17q21 leading to the formation of fusion proteins involving retinoic acid receptor α. Notably, the fusion protein interacts with p38 kinase and modulates its activity. Our findings imply that modulation of p38 by the APL fusion protein leads to increased arsenic trioxide uptake via aquaglyceroporin 9 in APL cells. This mechanism may account for the selectivity of arsenic trioxide for APL cells.
A Novel Bcr-Abl Mediated Pro-Survival Pathway: Reduction of Releasable Calcium Levels in the Endoplasmic Reticulum Inhibits Calcium Dependent Apoptotic Signaling.
Authors: Vejda Susanne , Piwocka Katarzyna , Cotter Thomas G. , O’Sullivan Gerald C. , McKenna Sharon L. 
Published: October 2019

Abstract
The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis (BC) and that this plays significant role in the achievement of drug resistance. We have established cell lines expressing low (C2 cells) and high levels (C4 cells) of Bcr-Abl to study the molecular mechanisms involved in disease progression especially with regard to drug resistance. Microarray analysis revealed that Bcr-Abl expressing cells exhibit increased expression of various endoplasmic reticulum (ER) resident proteins indicating that the ER is a novel target of Bcr-Abl. Recently, this organelle has been described as an important apoptotic gateway. Indeed, by measuring calcium levels (steady state and following drug treatment) in the ER and cytoplasm we could elucidate that the calcium dependent apoptotic signaling is disturbed in Bcr-Abl transformed cells. Moreover, inhibition of Bcr-Abl activity by Imatinib reversed this phenotype indicating that Bcr-Abl is directly modulating calcium homeostasis by yet unknown mechanisms. Two Bcr-Abl expressing human cell lines, K562 and BV173, also showed reduced amounts of calcium in the ER compared to the Bcr-Abl negative cell lines HL60 and Jurkat. Etoposide was chosen for apoptosis induction as this drug was classified to target not only the mitochondria (classical mitochondrial pathway) but also the ER (calcium dependent apoptotic pathway). Cell death was induced in the parental and Bcr-Abl transformed cell lines and calcium changes in the cytosol and the mitochondria were measured using organelle specific calcium probes. In C2 and C4 cells an early mitochondrial calcium overload was absent possibly due to the decreased amount of free releasable calcium in the ER. Moreover, other ER/calcium activated pathways were greatly diminished, such as cytosolic calcium elevation and calpain activation during apoptosis. It has been shown that decreased levels of releasable calcium in the ER is associated with cell survival. It is possible therefore that Bcr-Abl may exert some of its pro-survival or anti-apoptotic effects at the ER, by directly influencing the levels of releasable calcium. This study demonstrates a novel downstream consequence of Bcr-Abl signaling. The ability to negate calcium dependent apoptotic signaling is likely to be a major pro-survival mechanism in Bcr-Abl transformed cells.
Monoclonal Antibodies to a VWF-A2 Decapeptide with the C-Terminal Residue Tyr1605, Generated by ADAMTS13 Cleavage, Develop a Highly Sensitive ELISA for Its Activity and Characterize Upshaw-Schulman Syndrome.
Authors: Kato Seiji , Matsumoto Masanori , Matsuyama Tomomi , Hiura Hisahide , Fujimura Yoshihiro 
Published: October 2019

Abstract
We prepared murine monoclonal antibodies (mAbs) against a decapeptide of the VWF-A2 domain that has a C-terminal Tyr1605, generated by ADAMTS13 cleavage. These mAbs reacted neither with purified VWF nor the pentadecapeptide 1596-DREQAPNLVYMVTGN-1610, unless the Y1605-M1606 bond was cleaved. Using one of these mAbs, we developed a novel and highly sensitive ELISA for ADAMTS13 activity. In this assay, a recombinant VWF polypeptide (D1596–R1668) tagged with GST and His at the ends (GST-VWF73-His), is used as a substrate; upon cleavage by ADAMTS13, two fragments, GST-VWF10 and VWF63-His, are generated. Thus, the amount of GST-VWF10 can be directly measured using our mAb labeled with peroxidase. The specificity of this assay was confirmed by inhibition with EDTA, IgGs purified from thrombotic thrombocytopenic purpura (TTP) patients, and an anti-ADAMTS13 mAb. The detection limit of this assay was 0.5% of the normal.
Of 20 patients with congenital TTP (Upshaw-Schulman syndrome, USS) whose ADAMTS13 gene mutations were identified, 14 had a severe deficiency (<0.5%), and 6 had a moderate deficiency (0.55~1.3%) of ADAMTS13 activity. No correlation was found between these two groups in terms of the onset of clinical manifestations. Further, a unique asymptomatic carrier of USS with two mutations (R268P/P475S) in the gene showed 4.2 % of normal plasma ADAMTS13 activity in this novel ELISA, indicating that plasma levels of ADAMTS13 activity between 1.3 and 4.2 % represent a gray zone which may lead to clinical manifestations of TTP. Table 1 shows a clinical and laboratory data of USS patients included in this study.
Table 1. Clinical and laboratory data of 20 USS patients
Plasma Fibronectin Enhances Adhesion and Aggregation of Platelets by Both Its Incorporation into Adherent Platelets and Its Crosslinking to Fibrin Networks.
Authors: Cho Jaehyung , Mosher Deane F. 
Published: October 2019

Abstract
Plasma fibronectin has recently been demonstrated to stabilize growth of platelet-rich thrombi in injured mesenteric arterioles in vivo. We have determined the impact of two forms of insolubilized plasma fibronectin on adhesion and aggregation of platelets - fibronectin-crosslinked fibrin by activated factor XIII (fibronectin-fibrin) and fibronectin assembled by adherent platelets. In static adhesion assays, 2-fold more platelets adhered to fibronectin-fibrin than to fibrin. Antibody blocking studies demonstrated that a different mix of αIIbβ3, αvβ3, and α5β1 integrins mediate platelet adhesion to both substrates. At a shear rate of 300 or 1,250s-1 in a parallel plate flow chamber, more platelets adhered and aggregated on surfaces coated with fibronectin-fibrin than surfaces coated with fibrin. The number of adherent platelets and the volume of platelet thrombi increased by 3- to 4-fold when soluble fibronectin was co-perfused with platelets, which resulted in assembly of fibronectin by adherent platelets. Increased adhesion and aggregation of platelets were dependent on the amounts of fibronectin crosslinked to fibrin prior to perfusion or included in the perfusate. The effects of perfused fibronectin were blocked by two inhibitors of fibronectin assembly, the N-terminal 70-kD fragment of fibronectin or the functional upstream domain of protein F1 of Streptococcus pyogenes, indicating that assembly of soluble fibronectin by adherent platelets is mediated by the binding of the N-terminal region of fibronectin to the platelet surface. Thus, plasma fibronectin has the potential to enhance adhesion and aggregation of platelets in vivo as a result of being crosslinked to fibrin networks and its subsequent assembly by adherent platelets.
Interaction of High Molecular Weight Kininogen with Endothelial Cell Receptors suPAR, gC1qR and Cytokeratin 1 by Surface Plasmon Resonance (BiaCore).
Authors: Espinola Ricardo G. , Pixley Robin A. , Ghebrehiwet Berhane , Joseph Kusumam , Kuo Alice , Cines Douglas B. , Colman Robert W. 
Published: October 2019

Abstract
The physiologic activation of the plasma kallikrein kinin system (KKS) requires the assembly of these proteins on the cell membrane. High molecular weight kininogen (HK) binds to endothelial cells through an interaction with a multiprotein receptor complex that consist of: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR) and cytokeratin 1 (CK1). The affinity of HK and cleaved HK (Hka) for endothelial cells is KD=7–52 nM but the affinity for each of the three binding proteins is unknown. We first examined the direct binding of HK and Hka to the soluble receptor form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance (BiaCore). We linked suPAR, gC1qR and CK1 (800–1100 pg/mm2) by amine coupling to a CM-5 chip and perfused HK and Hka at a concentration ranging from 50 to 400 nM in the presence or absence of 10 μM ZnCl2. A Langmuir binding model with local fit (stoichiometry of 1:1) was used to analyse kon (association rate constant), koff (dissociation rate constant) and KD (equilibrium dissociation constant) for HK and Hka.
HK Hka KD kon koff KD kon koff Immobilized Protein nM M-1s-1 x104 s-1 x10-4 nM M-1s-1 x104 s-1 x10-4 gC1qR 0.8±0.7 12.3±5.0 0.8±0.5 0.7±0.5 24.4±10.9 1.4±0.5 CK1 15.1±1.0 3.6±0.3 5.4±0.7 6.3±1.9 5.8±0.3 3.6±0.9 suPAR 2313.5±1465.3 1.0±0.7 164.8±22.2 43.6±16.6 21.3±11.9 78.5±10.6
The binding of HK and Hka to the three receptors was zinc dependent. The affinity for HK and Hka was gC1qR>CK1>suPAR. The high affinity for gC1qR and CK1 was similar for HK and Hka and was due to a very slow koff. The affinity for Hka compared to HK was 2.5-fold tighter for CK1 but 50-fold tighter for suPAR. A reversed immobiization was then performed. We immobilized HK, Hka and LK (800 pg/mm2) and suPAR, gC1qR and CK1 were flowed over the chip at a concentration ranging from 50 to 700 nM. Only gC1qR bound to immobilized HK and Hka, with a KD of 6.31± 2.8 pM for HK and 2.86±1.5 pM for Hka while the other 2 receptors did not bind. Receptor integrity was shown by preincubating HK or Hka with the pure receptors and observing competition with the immobilized receptors. Among the HK/Hka receptors, complex formation was only observed between gC1qR and immobiized CK1 with or without Hka indicating that a CK1-gC1qR complex can form independently of kininogen. This study indicates that although suPAR has the weakest affinity of the three receptors it is the only one that distinguishes between HK and Hka. Only one binary complex was revealed by this technique between CK1 and gC1qR. These results lay the foundation for anlayzing the affinity of these receptors in a cellular environment.