bluuebunny/crossref_metadata_2025_split huggingface dataset:
Early Growth Response Factor EGR-1 Regulates Gαq Gene in Megakaryocytic Cells.
Authors: Jalagadugula Gauthami , Danny Dhanasekaran N. , Soochong Kim , Kunapuli Satya P. , Rao A. Koneti 
Published: October 2019

Abstract
Gαq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. We studied Gαq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic (MK) transformation. RT-PCR analysis of HEL cell RNA revealed that Gαq mRNA was relatively low in untreated cells and it increased after PMA treatment with a peak at 5 h. Immunoblot analysis of HEL lysates showed enhanced Gαq expression with PMA. Luciferase reporter gene studies on full length construct (upto −1116 bp from ATG) and its serial 5′ truncations revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. In the region −238/−202 consensus sites were noted for two transcription factors PU.1 and GATA-1 that are known to regulate several megakaryocytic genes. Deletions of these sites alone or together revealed no change in the transcriptional activity of the gene in reporter studies. The positive region −203/−138 contained two overlapping Sp1/AP-2/EGR-1 consensus sites at −202/−189 and −164/−150. Gel shift studies were performed on oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts. Protein binding occured with Gαq oligonucleotides 1 and 2, which was competed with excess unlabeled oligos but not by unlabeled Sp1 or AP-2 consensus oligos. Supershift assay using antibody against Sp1 revealed neither competition nor supershift, suggesting that Sp1 does not bind to these oligonucleotides. No protein binding was noted when Gαq oligos 1 or 2 were incubated with extracts known to contain Sp1 or AP-2. These results indicate that Sp1 and AP-2 do not bind to the Gαq oligonucleotide regions. Protein binding to oligonucleotides 1 and 2 was abolished by excess unlabeled consensus EGR-1 oligo, and by immunodepletion of the EGR-1 protein from the nuclear extract with anti-EGR-1 antibody. These experiments reveal that EGR-1 binds to both Gαq oligonucleotides −203/−175 and −174/−152. In luciferase reporter studies mutations in EGR-1 sites present in both oligonucleotides 1 and 2 markedly decreased gene activity indicating functional relevance. In further studies, reduction in endogenous EGR-1 expression with antisense oligonucleotide to EGR-1 inhibited PMA induced Gαq transcription and protein in HEL cells. Lastly, EGR-1 deficient mouse platelets also showed ~50% reduction in the Gαq protein (immunoblotting) relative to wild type platelets. These studies suggest that Gαq gene is regulated during PMA induced differentiation by EGR-1, a transcription factor that regulates a wide array of genes involved in cellular proliferation, differentiation, and apoptosis, and in vascular response to injury and atherosclerosis.
Shedding of Procoagulant Microparticles from Platelets through Integrin Outside-In Signaling Involving WASP and the Actin Cytoskeleton.
Authors: Cauwenberghs Sandra , Feijge Marion A.H. , Harper Alan G.H. , Sage Stewart O. , van Pampus Elisabeth , Curvers Joyce , Heemskerk Johan W.M. 
Published: October 2019

Abstract
Platelet activation by potent calcium-mobilizing agents, like collagen and thrombin or ionomycin, results in Ca2+- and calpain-dependent shedding of microparticles (MP) that are active in coagulation. Here, we describe and characterize a different mechanism of MP formation by platelets under storage in the absence of agonists. This shedding relies on integrin αIIbβ3 activity, since clinically used integrin blockers, like tirofiban or abciximab, reduce storage-induced MP shedding by 83±5.7% (p=0.006); inhibition of intracellular integrin signaling via Src kinase (PP1) or phosphoinositide 3-kinase (PI-3 kinase, wortmannin) inhibits this process by 52.3±14.3% (p=0.035) and 39±7% (p=0.005), respectively. Calpain inhibition suppresses MP formation by only 35±3.8% (p=0.003), while calpain activity is also not increased during storage. Levels of cytosolic Ca2+ concentration remain at 25–30 nM in the stored platelets. Treatment of platelets with recombinant WASP (Wiskott-Aldrich Syndrome Protein) peptide, activating the Arp2/3 complex and promoting actin nucleation, affects storage-induced MP shedding (p=0.001). Rho and Rac1, belonging to the family of Rho GTP-binding proteins, are involved in actin cytoskeleton regulation and platelet spreading. However, these GTP-binding proteins do not seem to be involved in MP shedding, as treatment of platelets with recombinant Rac1 peptide or with Rho kinase inhibitor is ineffective, as in the absence of these GTP-binding proteins. Furthermore, incubation of platelets with cytochalasin D, latrunculin B or jasplakinolide, which compounds affect actin filament polymerization, increases the amount of procoagulant MP (p<0.032). Interestingly, this effect is greatly reverted in the presence of integrin blockers or wortmannin. We determine that coagulation-active MP are abundantly present in stored platelet preparations that are used for transfusion purposes. The contribution of these MP to the coagulation process was studied after transfusion of these preparations to thrombocytopenic patients. Thrombin generation measurements indicate that the transfused MP initiate up to 25 nM concentrations of thrombin, which potentiate the procoagulant effect of platelets in these patients. Taken together, we conclude that the process of integrin-mediated MP shedding from apparently resting platelets is a uniquely regulated cellular response that operates independently of Ca2+ elevation and calpain activation, and contributes to coagulant activity in a clinically relevant way. It involves αIIbβ3 outside-in signaling, leading to WASP-mediated destabilization of the actin cytoskeleton, but independently of Rho and Rac1.
Integrin Linked Kinase Is Expressed in Acute Myeloid Leukemia Blasts and Its Inhibition Is Cytotoxic to Leukemic Progenitors.
Authors: Muranyi Andrew , Dedhar Shokat , Hogge Donna E. 
Published: October 2019

Abstract
Constitutive activation of the phosphatidylinositol-3-kinase (PI-3K) pathway is frequent in acute myeloid leukemia (AML) blasts and down-regulation of this pathway results in apoptotic death of these cells from many patient samples. Integrin linked kinase (ILK) is stimulated by activated PI-3K and ILK, in turn, activates AKT, a key downstream effector of the PI-3K pathway. The expression and activity of ILK are increased in a range of solid tumors. Small-molecule inhibitors of ILK activity have been identified and shown to inhibit tumour growth, invasion and angiogenesis. We investigated the possible role of ILK in AML blast and colony forming cell (AML-CFC) survival. Using Western blotting, ILK protein was detected in 30 of 35 primary AML blast samples although the levels seen were variable. ILK kinase activity correlated strongly with ILK protein expression as quantitated by densitometry (r = 0.91) in 6 samples where both were studied. Activation of the PI-3K pathway, as measured by detection of AKT phosphorylation on serine 473 (ser473), was also found in 27 of 30 samples expressing ILK protein. QLT0267 is a potent second generation small molecule inhibitor (from QLT Inc., Vancouver, Canada) which selectively inhibits the kinase activity of ILK but not a variety of other kinases. Treatment of AML blasts with QLT0267 resulted in a time and dose dependent decrease in p-AKT ser473 expression as well as downstream targets of AKT (p-S6, p-GSK-3β). 27 AML and 5 normal bone marrow (NBM) samples were incubated for 48h with QLT0267 or 10 μM of the PI-3K inhibitor, LY294002, and then plated in CFC assay. There was a direct correlation between the % AML-CFC kill seen with LY204002 and QLT0267 (r= 0.70 and 0.60 comparing % kill with LY294002 and QLT0267 at 3 and 10 μM, respectively). The IC50 of QLT0267 was ≤ 3μM for 6 of 23 AML samples and 0 of 5 NBM samples (range % kill of AML and normal CFC after treatment with 3μM; 0 – 86% and 0 – 23%, respectively). The IC90 for this inhibitor was ≤ 10μM for 9 of 27 AML and 0 of 5 NBMs (range % kill 6 – 99 and 30 – 68, respectively, for AML and normal CFC treated with 10μM QLT0267). Interestingly, AML-CFC from 4 AML samples in which ILK protein could not be detected were resistant to killing with QLT0267. Thus, ILK is expressed in a large proportion of AML samples. Inhibition of ILK kinase is cytotoxic to leukemic progenitors suggesting that this molecule is important for the survival of these cells. Approximately one third of AML samples tested were more susceptible to killing by ILK inhibition than NBM cells suggesting that selective targeting of malignant rather than normal hematopoietic cells may be achieved in some cases. Furthermore, it may be possible to predict which AML samples will be sensitive to inhibitors such as QLT0267 by measuring expression of the target protein.
Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells.
Authors: Okazaki Yasumasa , Yin Hong , Ma Yuxiang , Yeh Mary , Yeh Kwo-yih , Glass Jonathan 
Published: October 2019

Abstract
The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood, Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.
Characterization of NPM1-Mutated/FLT3 ITD-Negative Acute Myeloid Leukemia with Normal Karyotype by Gene Expression Profiling.
Authors: Bullinger Lars , Dohner Konstanze , Kranz Raphael , Rucker Frank G. , Frohling Stefan , Schlenk Richard F. , Pollack Jonathan R. , Dohner Hartmut 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) with normal karyotype comprises a large number of molecularly distinct variants. For example the presence of internal tandem duplications (ITDs) of the FLT3 (fms-related tyrosine kinase 3) gene is associated with poor outcome, whereas mutations of the NPM1 (nucleophosmin) gene are prognostically favorable. However, this effect is mainly attributed to the NPM1-mutated/FLT3 ITD-negative AML cases. While NPM1-mutated cases are characterized by a distinct gene expression pattern, it remains unclear whether NPM1-mutated/FLT3 ITD-negative cases also display a characteristic signature, which might provide additional insights into the molecular basis for the good clinical outcome. Thus, we sought to identify a molecular profile for AML cases with NPM1-mutated/FLT3 ITD-negative normal karyotype disease. Towards this goal, we profiled gene expression of 138 samples of adult AML patients with normal karyotype using DNA microarray technology. All samples analyzed were derived from AML patients entered within the randomized multicenter treatment trial HD-98A of the German-Austrian AML Study Group (AMLSG). Based on supervised data analyses we were able to identify a 116-genes comprising expression pattern correlated with NPM1-mutated and FLT3 ITD-negative AML cases. In accordance with previous findings in NPM1-mutated cases (Alcalay et al. 2005, Verhaak et al. 2005), the NPM1-mutated/FLT3 ITD-negative pattern was also in part characterized by a prominent HOX gene cluster, which clearly separated the NPM1-wildtype from the NPM1-mutated cases. Similarly, the expression levels of BAALC and MN1 were correlated with the NPM1 mutational status, with NPM1-unmutated cases displaying higher BAALC and MN1 expression in our data set. However, as expected the newly defined signature also defined a NPM1-mutated group that did not contain many FLT3 ITD-positive samples. This group was characterized by several interesting genes including for example TLE1, which encodes a Groucho/TLE family protein. Groucho/TLE family proteins are transcriptional co-repressors, which mediate repression essential in embryonic development and are involved in regulation of Wnt signaling in adult tissue. Moreover, we identified several other genes of potential pathogenic relevance which also have been previously shown to be predictive in normal karyotype AML. Our findings support a distinct molecular mechanism associated with the favorable outcome of NPM1-mutated/FLT3 ITD-negative AML cases. Furthermore, the reported signature might contribute to improved risk stratification and clinical management of AML patients with normal karyotype disease.
High Oxygen Environment during Pregnancy Rescues Sickle Cell Animia Mouse from Prenatal Death.
Authors: Ye Lin , Chang Judy C. , Chan Kalee , Lu Ronghua , Kan Yuet Wai 
Published: October 2019

Abstract
Several mouse models for sickle cell disease have been developed for the studies of the pathophysiology of sickle cell disease and the investigations of drug and gene therapies. In previous years, we have also succeeded in producing a sickle cell anemia mouse model. In this model the endogenous mouse α and β globin genes were knocked out and replaced by the human α and βs globin transgenes. The βs globin gene is contained in a 240kb YAC that preserves the entire native genomic context of the β-globin locus. These mice have anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, their γ globin switches to βs globin expression from around 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death we investigated the effect of placing the pregnant mothers into a high O2 environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O2 and kept the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from 0.2% to 32%. Moreover, 55% to 88% of the newborn sickle cell anemia mice survived in the oxygen treated group, whereas none of the sickle cell anemia mice survived in regular air breeding conditions. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs by histological analysis. These pathological finding are very similar to those find in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice will facilitate physiological and therapeutic studies of sickle cell anemia.
Contribution of Coagulation Pathways and Fibrinolysis in Generation of Kinins.
Authors: Eve Moreau Marie , Désormeaux Anik , Fortin Jean-Philippe , Rivard Georges E. , Lepage Yves , Brown Nancy J. , Gauthier Francis , Marceau François , Adam Albert 
Published: October 2019

Abstract
BACKGROUND. Classically, there are two main pathways by which kinins are generated: the plasma and tissue kallikrein-kinin systems. Each of this complex multi-protein system includes tissue or plasma kallikrein, which generate kinins from high or low molecular weight kininogen. In addition to plasma and tissue kallikreins, other serine proteases may have a kinin-forming capacity.
OBJECTIVES. The main purpose of this study was to characterize the kininogenase activity of plasma kallikrein, thrombin (factor IIa), factor Xa, and plasmin in a purified system. We also aimed at characterizing the kinetic profile of generation of kinins in a whole blood system where coagulation and fibrinolysis were monitored by thromboelastography.
METHODS. Bradykinin (BK) released from both low and high molecular weight kininogens and quantified with a specific competitive enzyme immunoassay was used to calculate the kinetic parameters for the different kininogenase activities. Physicochemical and pharmacological characterization of immunoreactive kinins confirm their nature. The kinetic profile of kinin generation was studied during the process of coagulation which was initiated with kaolin or with tissue factor and monitored by thromboelastography.
RESULTS. BK was released not only by plasma kallikrein; both plasmin and factor Xa hydrolyzed high molecular kininogen to produce BK. In the case of thrombin, the release of both BK and kallidin was detected from low and high molecular weight kininogen. When applied to whole blood, this analytical approach indicated that the kinin-forming capacity of the coagulation intrinsic pathway was higher than that of the extrinsic pathway. The triggering of fibrinolysis by tissue plasminogen activator dramatically potentiated the release of des-Arg9-BK.
CONCLUSIONS. Although plasma kallikrein is considered as being the main kinin-forming enzyme, other serine proteases of the coagulation and the fibrinolysis pathways are able to generate kinins and could play a role in different pathologies where kinins are potentially involved.
Amelioration of Inflammation, Angiogenesis and CTGF Expression in an Arthritis Model by a TSP1-Derived Peptide Treatment.
Authors: Rico Mario C. , Castaneda Julian L. , Manns Joanne M. , Uknis Audrey B. , Safadi Fayez F. , Popoff Steven N. , De La Cadena Raul A. 
Published: October 2019

Abstract
Objective. To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis.
Methods. Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-APS). Animals were divided into four groups:negative control and groups receivingno treatment,treatment with a TSP1-derived peptide andtreatment with a scrambled peptide.
Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR.
Results. Histological data indicated that TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunohistochemical and RT-PCR analyses showed an increase of the CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to TSP1-derived peptide treated animals. By immunohistochemistry, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals.
Conclusion. Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.
Vasculature measurements at the perisynovial tissue of the TSP1-derived peptide treated (DT) and scramble peptide treated (DS) animals after day 16 PG-APS-induced erosive arthritis. Vascular Measurements Disease TSP1-derived peptide treated (DT) Disease scrambled peptide treated (DS) P Value Vascular area mean±SEM (μm2) 77.41 ± 7.96 μm2 336 ± 49.21 μm2 p=0.0008 Vessel wall thickness (μm) 3.53 ± 0.21μm 8.92 ± 0.48;μm p<0.0001 Vascular index (VI) ± SEM (VI=total number of vessels/mm2) 5.6x10−3 ± 0.37x10−3 vessels/mm2 17.9x10−3 ± 1.33x10−3 vessels/mm2 p=0.006
Human Embryonic Stem Cell-Derived “NET-Like” Structure Serves as a Hematopoietic Progenitor Niche and Favors Generation of Mature Megakaryocytes and Functional Platelets.
Authors: Takayama Naoya , Nishikii Hidekazu , Hiroyama Takashi , Nakamura Yukio , Tsukui Hiroko , Eto Koji , Nakauchi Hiromitsu 
Published: October 2019

Abstract
Human embryonic stem cells (hESCs) are proposed as an alternative source in regenerative medicine while being a promising tool to trace earliest ontogeny in vivo. Anucleate platelets can be irradiated to eliminate residual ESCs that potentially cause immunological rejection and teratoma formation in transplantation. Herein, we describe for the first time the generation of functional platelets from hESCs (Kyoto hES1, -2, and -3) by a novel method that is distinct from previously established OP9 co-culture. Exogenous combination of TPO, VEGF and IGF-II allowed hECSs to facilitate the differentiating kinetics of hematopoietic marker emergence when cultured on feeder cells, 10T1/2, whereas independent single administration had little effect on hematopoietic differentiation (P<0.01). Consequently, unique “NET-like” structures expressing VE-cadherin low+, PECAM-1 high+, CD34 high+, CD45 + and VEGF-R2 + were observed around 14–15 days of culture. These structures contained hematopoietic progenitors capable of forming hematopoietic colonies in semisolid culture, or of differentiating into mature megakaryocytes in the presence of TPO, IL-6, and IL-11 (around 45 % of total cells displayed GPIbα +GPIX + by day 23). While totally 24–25 days culture was required for production of hESC-derived integrin αIIb +GPIX + platelets in this way, the platelets exhibited specific fibrinogen-bound to integrin αIIbβ3 in the presence of G-protein coupled receptor agonists in a dose-dependent manner. The efficiency of platelet production appeared to be well correlated with the frequency of “NET-like” structure. Thus, this structure may be a useful hallmark in our newly obtained culture method that may serve as an invaluable tool for not only generation of hESC-derived functional platelets but also studying niche machinery in hematopoiesis.