bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Genome-Modified Mouse Line with an Insertion Mutation at the AML1/Runx1 Gene Locus Detected from a Patient with Myelodysplastic Syndrome.
Authors: Fukushime-Nakase Yoko , Nakao Mitsushige , Horiike Shigeo , Nishimura Motohiro , Taniwaki Masafumi , Sugimoto Tohru , Okuda Tsukasa 
Published: April 2020

Abstract
AML1 (Acute Myeloid Leukemia 1; also known as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays important roles in initiating definitive hematopoiesis, thymocyte development, and platelet production. AML1’s DNA-binding is mediated through its Runt domain, and their further association with the non-DNA-binding subunit, CBFβ stabilizes the resultant ternary complex to be functional. AML1 is frequently involved in leukemia-associated chromosome translocations, through which AML1 fusion-proteins with strong dominant-negative effects to normal CBF are generated, thus contributing to the leukemic transformation. Recently, genomic mutations of the AML1 gene locus have been described to associate with cases of acute myeloblastic leukemia and myelodysplastic syndromes (MDS), and pedigrees of familial platelet disorder with propensity to develop acute myeloblastic leukemia (FPD/AML). Frequent sites of the point-mutations are confined to the residues within the Runt domain, such as, R139, R174, or R177, which are important for the DNA-contact of the molecule. Preliminary results from studies using knockin mice carrying mis-sense mutations for these residues so far revealed that the mutants are biologically inactive when homozygously inherited. However, the mechanisms how the mutations contribute to the onset of the leukemia remain largely to be elucidated. In order to further characterize the aberration of AML1, we generated another genome-modified mouse line which harbors an insertion mutation of this gene locus. As reported previously, we identified a case of refractory anemia with excess of blasts (RAEB) whose blast cells have a mutated allele where three base-pair, ATC, is inserted after codon 150 (I150ins) of the molecules. By this mutation, an additional isoleucine residue is inserted into the 10th β-strand of the Runt domain sequences which provides a facet to interact with CBFβ. We introduced the cDNA of I150ins into AML1/Runx1 gene locus by means of a gene-knockin approach in mouse ES cells, and transmitted the allele into mouse germline through conventional methods. Subsequent heterozygous mice were viable and fertile. In contrast, homozygous mice die in utero similarly to the mice with simple disruption of this gene. These results indicate that this insertion mutation leads to the complete loss of its biologic function as were observed for the point-mutant mice. Further careful examinations of the heterozygous mice for this mutant allele in comparison with those for point-mutation animals and simple haplo-insufficient ones will provide valuable insights into the molecular mechanisms of AML1-related leukemic transformation.
Species-Specific Differences in Intracellular Transport Reveal a Golgi-Localization Requirement for D816V Mutant Kit Receptor To Induce Leukemia.
Authors: Xiang Zhifu , Cain Jennifer , Kreisel Frederike , Tomasson Michael H. 
Published: April 2020

Abstract
Mutations in the c-Kit gene (KIT) that result in constitutive tyrosine kinase activity are associated with mastocytosis/mast cell leukemia, gastrointestinal stromal tumors (GIST), and acute myeloid leukemia (AML). Expression of a murine Kit allele with a mutation in the phosphotransferase domain (Kit D814V) renders murine Ba/F3 cells factor-independent and causes lymphocytic leukemia in mice. An animal model using the homologous human allele (KIT D816V) has not been described. We generated retroviral expression constructs using wild-type and mutant human KIT and murine Kit that achieved equivalent protein levels in human-derived 293T cells. When expressed in murine cells however, despite equivalent transcript levels, the KITD816V protein specifically was present in lower abundance, and unlike the murine mutant, the human D816V was unable to transform Ba/F3 cells without prior selection. In contrast, human KITD816V rapidly transformed human TF-1 cells to factor independence. Fusion of the extracellular and transmembrane domains of murine Kit to the intracellular domain of human KIT (Hybrid KIT WT and Hybrid KITD816V) facilitated higher protein expression in murine cells, and rapid transformation of both Ba/F3 and TF-1 cells. Endoglycosidase digestion revealed that both human and murine mutant Kit receptors were incompletely glycosylated, and by immunohistochemistry we found that the murine and hybrid mutant KIT receptors were predominantly localized to the golgi. However, the human KIT D816V protein, in murine cells, failed to traffic past the endoplasmic reticulum (ER), while in human cells, KITD816V was able to traffic through the ER to the golgi. In murine cells expressing human KITD816V, downstream signaling pathways, including Stat3, Stat5 and Akt were activated to a lesser extent compared to cells expressing the murine KitD814V or hybrid KITD816V. Treatment of cells with small molecule inhibitors of protein trafficking demonstrated that activation of downstream signaling pathways activated by Hybrid KITD816V required golgi but not cell membrane localization. Lastly, mice transplanted with bone marrow transduced with the human KITD816V remained free of disease while mice transplanted with the golgi-localized Hybrid KITD816V succumbed to fatal myeloproliferative disease (MPD) with 100% penetrance. Taken together, our data demonstrate that murine and human cells traffic the KITD816V protein differently and that leukemogenic signals require localization of the mutant receptor to the golgi apparatus. This is the first report of an animal model of myeloid leukemia/MPD using the KIT oncogene and will be useful for elucidating the mechanisms by which KITD816V contributes to leukemogenesis and for testing novel therapeutic strategies.
Deregulation of Retinoic Inducible Gene---Rig-I Underlies the Pathogenesis of Acute Promyelocytic Leukemia.
Authors: Zhu Jiang , Shen Shu-Hong , Chen Li-Juan , Huang Qiu-Hua , Zhang Nan-Nan , Sun Yi-Wu , Emerson Stephen G. , Chen Sai-Juan , Wang Zhu-Gang , Chen Zhu 
Published: April 2020

Abstract
numerous chromosomal translocations, all of which involve the gene encoding retinoid acid receptor a (RARa), resulting in the generation of corresponding oncogenic fusion proteins including PML-RARa and PLZF- RARa (hereafter referred as to X- RARa). However, through deregulating which downstream genetic program(s) X- RARas perturb the hematopoiesis has remained unclear. Here we report that Rig-I, a putative RNA helicase, was identified in RA-induced granulocytic differentiation of APL cell line NB4 cells. Interestingly, the up-regulation of Rig-I was also realized in RA-treated freshly isolated primary APL blasts harboring PML-RARa and HL-60 cell line, but not in RA-treated NB4 variant NB4-R2 (RA refractory) and APL blasts carrying PLZF-RARa, suggesting that the induced Rig-I activity is one essential step underlying the granulocytic differentiation of myeloid leukemia cells. In line with this, the knockout mice of Rig-I produced a phenotype of profound granulocytosis with moderate differentiation blockage; and conversely the overexpression of Rig-I inhibited in vivo hematopoietic reconstitution of transduced normal primitive bone marrow cells. Most importantly, the Rig-I expression was found significantly repressed in myeloid compartment of X-RARa transgenic mice, and that the retroviral transduction of Rig-I cDNA into murine APL blasts carrying hMRP8-PML/RARa greatly decreased their leukemogenesis reconstitution ability in sublethally-irradiated syngeneic recipients. Moreover, we found that Rig-I integrity is required for the full expression of TRAIL, whose expression is critical factor in determining the pool size of myeloid cells. Such, we characterize Rig-I as a crucial negative regulator of granulopoiesis and a potential candidate for developing molecule-based target therapy of myeloid leukemia.
Role of EVI1 in Cell Cycle Regulation: Relevance of Specific Target Genes.
Authors: Perkins Archibald S. , Xiao Ying-Yi , Lezon-Geyda Kimberly , Lin Sharon , Camargo Fernando , Tuck David , Yatsula Bogdan 
Published: April 2020

Abstract
Mds1-Evi1 is a complex locus with two distinct transcriptional start sites, four possible polyadenylation sites, and nineteen exons spread over half a megabase that encodes at least six protein isoforms containing variable numbers of DNA-binding zinc finger motifs. In mouse and some other species, the locus is a common site of proviral insertion in myeloid leukemia, while in human it is a site of nonrandom chromosomal rearrangement in myelodysplasia, AML, and CML blast crisis. One such translocation is t(3;21), encoding the AML1-MDS1-EVI1 (AME) fusion protein. We have characterized the RNA transcripts and proteins generated by Mds1-Evi1 in murine leukemic cells bearing Evi1 proviral insertion using three different antisera specific for the N-terminal and C-terminal domains of EVI1, as well as MDS1, and find consistent overexpression of three protein isoforms of 135, 123, and 103 kDa (EVI1A, B, and C, respectively) while other isoforms, including MDS1-EVI1, are not normally expressed. However, by knocking lacZ into the Mds1 first exon we showed that it is normally expressed in hematopoietic progenitors, similar to Evi1. The N-terminal zinc finger domain present in the EVI1A and B isoforms binds to the GACAAGATA motif with high affinity, and single missense mutations in zinc finger six abrogate high affinity binding. Using two different bioassays, we have demonstrated that cellular transformation by either the EVI1A isoform or by AME is dependent on DNA binding via this N-terminal domain, thus indicating that transformation is dependent on the action of this domain on specific target genes. To determine the biological effect of Evi1 on leukemic cell growth, short hairpin RNAs were used to suppress Evi1, and this resulted in slowing of the cell cycle and prolongation of the G2/M phase. RNA expression profiling revealed significant changes in gene expression accompanied Evi1 suppression, including some involved in cell cycle control. Upon long term Evi1 suppression, numerous markers of myeloid differentiation were induced, indicating the acquisition of a differentiation-like phenotype. These data indicate an effect of EVI1 on both cell cycle and differentiation, and suggest that the effect on cell cycle is direct. To gain further insight into the nature of EVI1 target genes, we utilized tet-regulated expression of Evi1A, and identified a set of putative targets of the A isoform that includes genes directly involved in cell cycle. However, the fold changes are relatively minor, and although the kinetics of activation suggests these are direct targets, this has not been conclusively demonstrated. We predict on the basis of these data that EVI1 expression modulates several key regulators of cell cycle and this results in an acceleration of cell cycle. With high level EVI1 expression, block to differentiation is induced as well.
Hyperhomocysteinemia in Relation to Total and Cardiovascular Death after Coronary Artery Bypass Grafting. A Prospective Study.
Authors: Girelli Domenico , Martinelli Nicola , Olivieri Oliviero , Trabetti Elisabetta , Pizzolo Francesca , Friso Simonetta , Bozzini Claudia , Tenuti Ilaria , Faccini Giovanni , Lotto Valentina , Pignatti Pier Franco , Mazzucco Alessandro , Corrocher Roberto 
Published: April 2020

Abstract
Background: Hyperhomocysteinemia (HHCY), a known risk factor for thrombosis, is frequently found in patients with chronic ischemic heart disease, but its independent prognostic impact is still controversial.
Aim: To evaluate the relationship between HHCY and mortality in patients after coronary artery bypass grafting (CABG) surgery.
Methods: We prospectively followed 353 patients (mean age 60.1 ± 9.0; 83.3% males) who underwent elective CABG between May 1996 and May 1999.
Results: after a median follow-up of 58 months, 36 patients (10.1%) had died, 28 because of cardiovascular events (Figure 1). Baseline homocysteine levels &gt;90th of distribution (i.e. &gt;25.2 μmol/L) were independently associated to total mortality: OR = 2.39, 95% CIs 1.02–5.59, P=0.035, after adjustment for age, sex, baseline renal function (glomerular filtration rate estimated by MDRD equation), and high-sensitivity C Reactive Protein, by logistic regression. HHCY was also independently associated with mortality due to cardiovascular events, adjusted as above including the presence or absence of myocardial infarction prior to CABG (OR = 2.66, 95% CIs 1.02–6.96, P=0.045).
Conclusions: Moderate (&gt;25.2 μmol/L) HHCY is an independent predictor of total and cardiovascular mortality after CABG. At least in this specific subgroup of cardiovascular patients, low-cost strategies to lower homocysteine (i.e. supplementation with folate and B vitamins) should be implemented.
Figure Figure
A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.
Authors: Canobbio Ilaria , Stefanini Lucia , Guidetti Gianni F. , Balduini Cesare , Torti Mauro 
Published: April 2020

Abstract
The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.
Detection and Mechanisms of GPIIb-IIIa Antagonist Induced Thrombocytopenia: Role for Antibody Induced Platelet Activation.
Authors: Linden Matthew D. , Frelinger Andrew L. , Furman Mark I. , Krueger Lori A. , Li Youfu , Fox Marsha L. , Hughes Annette , Lown John , Barnard Marc R. , Przyklenk Karin , Michelson Alan D. 
Published: April 2020

Abstract
After the administration of GPIIb-IIIa antagonists, 1 – 3% of patients become thrombocytopenic, and 0.5 – 1% of patients develop profound thrombocytopenia. This GPIIb-IIIa antagonist-induced thrombocytopenia may be antibody mediated. In this study we demonstrated that GPIIb-specific monoclonal antibody 5B12 augmented platelet activation, as determined by platelet surface P-selectin and leukocyte-platelet aggregation) in the presence of therapeutic concentrations of abciximab, but not epitifibatide or tirofiban. Furthermore, the GPIIIa-specific monoclonal antibody Y2/51 augmented platelet activation in the presence of therapeutic concentrations of eptifibatide or tirofiban, but not abciximab. This activation was abolished by an FcγRII receptor (CD32)-specific antibody and was absent when Fab fragments 5B12 or Y2/51 were used. These results suggest that (i) GPIIb-IIIa antagonists cause conformational changes in GPIIb-IIIa; resulting in (ii) antibody binding to GPIIb-IIIa; and, consequently (iii) platelet activation via the FcγRII receptor. To determine whether this mechanism is operative in vivo, plasma from patients who developed abciximab-induced thrombocytopenia (n=2), eptifibatide-induced thrombocytopenia (n=5) and normal donors (n=7) was incubated with ABO Rh matched donor whole blood with and without therapeutic concentrations of GPIIb-IIIa antagonists. Platelet activation was assessed by flow cytometric measurement of platelet surface P-selectin. Abciximab, but not eptifibatide or tirofiban, increased platelet surface P-selectin in patients who developed abciximab-induced thrombocytopenia. Eptifibatide, but not abciximab or tirofiban, increased platelet surface P-selectin in patients who developed eptifibatide-induced thrombocytopenia. Platelet surface P-selectin in normal donors was not changed by abciximab, tirofiban or eptifibatide. These data (i) provide novel insight into the mechanisms of GPIIb-IIIa antagonist induced thrombocytopenia; and (ii) suggest a possible assay for the clinical diagnosis and prediction of this phenomenon.
Platelet surface P-selectin after addition of a GPIIb-IIIa antagonist (% baseline fluorescence). *p&lt;0.05 Abciximab Eptifibatide Tirofiban Normal donors 103 105 99 Eptifibatide-induced thrombocytopenia 103 170 * 104 Abciximab-induced thrombocytopenia 1571 * 108 105
The 3-Dimensional Architecture of Platelets in the Native State by Electron Cryotomography.
Authors: Marsh Michael P. , Chang Juan , Chen Junmei , Lopez Jose , Chiu Wah 
Published: April 2020

Abstract
Conventional electron microscopy requires that samples be chemically fixed, embedded in plastic and stained prior to examination. This technique always raises uncertainty as to whether the observations are faithful to the native cellular organization. These limitations are overcome with the technique of electron cryomicroscopy, in which samples are prepared and observed in the frozen, hydrated state. A modification of this technique, electron cryotomography, is a method capable of recording images from the same specimen area at different tilt angles under low electron dose conditions, revealing the spatial relationships of organelles and molecular assemblies within a cell that has not been subjected to chemical fixation or metal staining.
Here, we used electron cryotomography to examine the 3-dimensional architecture of platelets in the resting state and in various stages of activation. Platelets in citrated plasma isolated from healthy human subjects were allowed to settle onto a perforated carbon grid and frozen immediately in liquid ethane or were allowed to settle for different time periods on a grid coated with fibrillar type 1 collagen before being frozen. The platelets suspended across holes were embedded in a thin layer of vitreous ice. Successive images of these frozen, hydrated platelets were then acquired as the stage was tilted through a range of ±70° using a JEM2010F electron cryomicroscope. Each image was recorded using a 4kx4k CCD camera. The digital images were computationally aligned and merged to reconstruct a 3-dimensional density map with an 4.0 gigapixel volume. Multiple tilt-series were reconstructed and analyzed using segmentation and visualization tools. The platelet reconstructions were annotated in terms of the sizes, shapes and relative locations of the subcellular components, including mitochondria, α and dense granules, dense clusters, the dense tubular system, microtubules, and the surface-connected canalicular system. We observed great variability in the extent of ordered structure in the α granules, and in the filopodia adhering to the grid surface. The tomograms resolve the fibrous actin bundles driving filopod extension and the individual actin cross-links within the bundles. In some platelets, we observed membrane tethers that attached to the surface but were devoid of actin. This represents the first 3-D visualization of human platelets under native conditions. These observations and the ease of generating such data suggest that electron cryotomography can become a useful tool for dissecting the molecular sequence of cytoskeletal rearrangements occurring during platelet activation and granule secretion and for understanding the structural basis of platelet diseases.