bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neither Free αIIb nor Free β3 Limits αIIbβ3 Biogenesis, and Pro-αIIbβ3 Adopts a Conformation Akin to Ligand-Bound Mature αIIbβ3.
Authors: Mitchell W. Beau , Newman Peter J. , Coller Barry S. 
Published: April 2020

Abstract
The first steps in the biogenesis of αIIbβ3 involve the individual synthesis of each subunit within the endoplasmic reticulum, followed by complex formation. Previous studies performed in HEL cells and in megakaryocyte-lineage cells cultured from cryopreserved progenitor cells of patients with CML have led to conflicting data with regard to whether αIIb or β3 is produced in limiting amounts that might then control complex formation. To study this question in greater detail, we conducted immunoprecipitation studies on HEK293 cells transfected with αIIb and β3 cDNAs and megakaryocyte-lineage cells derived from human umbilical cord blood grown in the presence of thrombopoietin and IL-11. Cells were labeled for 15 min with 35S-methionine/cysteine, and then lysed after 2 hr with a 1% triton X-100 buffer. The antibodies employed included murine monoclonal antibodies 10E5 (anti-αIIbβ3 complex, which, reacts with αIIb cap domain), 7E3 (anti-αIIbβ3 + αvβ3, which reacts with β3 specificity-determining loop and α1 helix), AP-5 (LIBS anti-β3, which reacts with β3 amino acids 1 - 6), 7H2 (anti-β3, which reacts with PSI domain near C13), 1990 (anti-αIIb, which reacts at unknown site), and LIBS2 (LIBS anti-β3, which reacts with a site within amino acids 602 – 690). The relative amounts of pro-αIIb, mature αIIb, and β3 immunoprecipitated by this panel of antibodies is shown in Table 1. Results were similar in both the HEK293 cells and in megakaryocyte-lineage cells derived from human umbilical cord blood. These data indicate that there are sizeable pools of both free pro-αIIb and free β3 in both cell types, and thus neither subunit controls complex formation by limited availability. Based on the selective precipitation of pro-αIIb + β3 by AP5, it appears that the pro-αIIbβ3 complex adopts a conformation similar to that of ligand-bound mature αIIbβ3. We conclude that αIIbβ3 complex formation is slow relative to the production of the individual subunits, and is probably controlled either by the ability of the subunits to adopt the proper fold and/or to interact with a chaperone(s) that facilitates complex formation. Assuming that AP5 selectively recognizes αIIbβ3 complexes in which the angle of the β3 βA (I-like) domain - hybrid domain interface results in separation of the αIIb and β3 leg domains, these data also suggest that αIIbβ3 head-head interactions precede αIIbβ3 leg-leg interactions during biogenesis, and that αIIb maturation occurs rapidly after αIIbβ3 leg-leg interactions. Such a model is consistent with our previous data demonstrating that pro-αIIb binds to the ER membrane-resident chaperone calnexin via the N-linked glycan at αIIb N15.
Table 1 Antibody Pro- αIIb Mature αIIb β3 1990 ++++ +++ +++ 10E5 ++ +++ ++ 7E3 ++ +++ ++ 7H2 ++ +++ ++++ LIBS2 + − ++++ AP5 ++ − ++++
Age-Decline in the Activity of the Plasma Membrane Ca2+ Pump (PMCA) of Normal Human Red Blood Cells (RBCs).
Authors: Bookchin Robert M. , Daw Nuala , Etzion Zipora , Tiffert Teresa , Lew Virgilio L. 
Published: April 2020

Abstract
We recently demonstrated that the activity of the PMCA varies greatly among the RBCs in a normal blood sample (Lew et al., Blood102:4206,2003). To test the possibility that these variations might be related to cell age, we designed a new experimental protocol to separate RBCs with different PMCA Vmax, which used glycosylated hemoglobin (Hb) A1c as an age marker for the normal RBCs, and avoided Co2+ (used in our usual PMCA activity assay) which we found to interfere with Hb A1c measurements: The ionophore A21837 was used to generate a high, rapid and uniform [CaT]i in RBCs, which were suspended in a 90mM-K+ buffer to prevent RBC dehydration by Gardos channel activation. These RBCs were then washed in high-K+ buffer (ice-cold to inhibit the Ca2+ pump) with 1% albumin to remove the ionophore. At t=0, the washed and packed ionophore-free cells were delivered into 20 volumes of high-K+ buffer at 37oC to initiate Ca2+ extrusion by the PMCA, and then sampled at 15 sec intervals into 25 vol of an ice-cold, K+-free, isotonic buffer containing 10 mM SCN−; these conditions were designed to trap un-extruded Ca2+ and to elicit rapid dehydration of those cells which had not yet pumped out all their Ca2+ load. After ~ 40 min at 0oC each sample was spun; the packed RBCs were resuspended in 10 vol of the same buffer and spun again through diethylphthalate oil (D=1.117 g/ml) to separate the dehydrated RBCs (pellets) from the non-dehydrated cells. The fraction of cells in pellets and on top of the oil was estimated in each sample from Hb measurements, and their Hb A1c content was measured by HPLC. Initially, almost all RBCs, except those with the most vigorous pumps, were recovered in the pellets, but with time, the fraction of cells which had fully extruded their Ca2+ load and were recovered on top of the oil approached 100%. The progressively smaller pellets contained RBCs with progressively weaker pumps; their Hb A1c fraction increased with decrease in the yield of RBCs in the pellet. This inverse correlation between yield and Hb A1c fraction in the pellets was observed in all six experiments performed with RBCs from four donors. Since the decline in PMCA activity correlated directly with an increase in Hb A1c, the observed population variation in PMCA activity reflects an age-related decline in PMCA activity. Whether this decline is due to progressive glycosylation of of a lysine residue near the catalytic ATP domain on the pump ATPase (Gonzalez Flecha et al., J Membr Biol171:25,1999) or to other mechanisms, remains to be determined. The age-decline in PMCA activity may be responsible for the increasing density of aging RBCs, by allowing intermittent episodes of [Ca2+]i elevations in the circulation, with transient Gardos channel activation and gradual dehydration by net KCl and water loss.
Alpha Hemoglobin Stabilizing Protein (AHSP) Is a Susceptibility Gene to Drug /Infection-Induced Hemolytic Anemia.
Authors: Kanno Hitoshi , Kamatani Naoyuki , Hamada Takako , Furihata Kaori , Hattori Yukio , Miwa Shiro , Fujii Hisaichi 
Published: April 2020

Abstract
Approximately 500 patients are diagnosed as having congenital hemolytic anemia every year in Japan, and the etiology in 75% of these patients involves red cell membrane disorders. The remaining cases are categorized as congenital non-spherocytic hemolytic anemia (CNSHA) and only about 25 % of CNSHA patients are diagnosed as either unstable hemoglobinopathies or erythroenzymopathies. As a result, up to 100 subjects remained diagnosed as having idiopathic hemolytic anemia. Recently, α-hemoglobin stabilizing protein (AHSP) has been identified, and found to play an important role as molecular chaperones in the stabilization of unstable free α-globin. Intact α-globin monomer generates reactive oxygen species, resulting in damage to red cell protein and lipids. AHSP knockout mice showed compensated hemolysis with Heinz bodies. Genetic defects in protection against oxidative stress such as glucose-6-phosphate dehydrogenase deficiency may account for acute hemolysis only when subjects are exposed to either oxidative drugs or infection, but chronic hemolysis is not evident in those cases. These observations led us to hypothesize that AHSP gene defects might account for either Heinz body-positive or drug/infection-induced hemolytic anemia.
We examined 25 CNSHA patients with unknown etiology: 6 cases showing Heinz body-positive red cells and 19 cases showing episodes of drug/infection-induced acute hemolysis. Erythroenzymopathies and unstable hemoglobinopathies were ruled out using red cell enzyme analysis, isopropanol stability tests and DNA sequencing of globin genes. Seven SNPs (single nucleotide polymorphisms) surrounding the AHSP gene (rs 4889672, rs 8046452, rs 8050390, rs 4296276, rs 17677, rs 10843, and a novel SNP at 92-bp upstream of rs 4889672) were examined, and compared with 100 control subjects. We found that a certain haplotype, 5′-GTCGA**-3′, was significantly prevalent in CNSHA cases (p=0.0086). Each SNP within the haplotype is located in the 5′-flanking region of AHSP gene, suggesting that the SNP(s) might account for decreased expression of AHSP gene. The reporter gene assay using K562 cells revealed that a construct containing 5′-GTCGA**-3′ had about 30% of transcriptional activity compared to a major haplotype 5′-ACCAG**-3′. Taken together, we conclude that the AHSP gene is correlated to disease-susceptibility in a group of hemolytic anemia with either Heinz body-positive or drug/infection-induced episodes.
A Pilot Study To Evaluate the Safety, Efficacy and Effects on Liver Function of Granulocyte Colony-Stimulating Factor To Mobilize Hematopoietic Stem Cells in Patients with Liver Cirrhosis.
Authors: Isidori Alessandro , Catani Lucia , Talarico Simona , Lorenzini Stefania , Loggi Elisabetta , Gramenzi Annagiulia , Bonifazi Francesca , Aluigi Michela , Bernardi Mauro , Andreone Pietro , Baccarani Michele , Lemoli Roberto M. 
Published: April 2020

Abstract
In animal models hematopoietic stem cells (HSCs) and the hematopoietic cytokine granulocyte colony-stimulating factor (G-CSF) contribute to tissue regeneration after acute or chronic liver damage. In this study, we assessed whether: 1) G-CSF can be safely administered to patients with liver cirrhosis to expand and mobilize HSCs into peripheral blood; 2) G-CSF treatment affects residual liver function. Eighteen patients with a Child-Turcotte-Pugh (CTP) score &lt; 10 and a Mayo Model for End Stage Liver Disease (MELD) score &lt; 20 were considered eligible for the study. Increasing doses of G-CSF were administered subcutaneously for 7 consecutive days to 5 cohorts of 3 patients each, starting from 2.2 μg/kg/daily, until the achievement of the maximum-tolerated dose and/or the successful mobilization of ≥10 CD34+ cells/μl in at least 2/3 patients. G-CSF mobilizing dose was found at 15 μg/kg/day. Four additional patients were treated with the mobilizing G-CSF dose. All of them showed a successful mobilization of HSCs with a median peak value of CD34+ and CD133+ cells of 27.1(±15.5) and 15.2 (±5.1)/μl, respectively. Circulating HSCs were then collected by single volume leukapheresis and a median of 1.4(±0.73)x106 CD34+ HSCs/kg body weight were cryopreserved. G-CSF treatment induced a significant increase of hepatocyte growth factor serum level. When we analyzed stem cell subsets, we found significant mobilization of early hematopoietic (CD34+/CD38−) and endothelial (CD34+/KDR+, CD133+/KDR+) progenitors as well as CD34+/CXCR4+ cells. No severe adverse events were observed at any dosage or during leukapheresis. According to CTP and MELD score, no significant modification of liver function was observed during the treatment period and follow up (median=198 days) in both mobilizing and non-mobilizing patients. Nevertheless, in all treated patients a significant reduction of alpha-fetoprotein values was observed (p&lt;0.001). In conclusion, the administration of G-CSF to patients with liver cirrhosis is safe, feasible and capable of mobilizing HSCs at the dosage of 15 μg/kg/day. Our study represents the first step for evaluating the role of G-CSF and mobilized HSCs to improve liver function in patients with liver cirrhosis.
Characterization of ‘Early’ vs ‘Late’ Endothelial Progenitor Cells (EPCs) Which Are Derived from Human Umbilical Cord Blood (HCB) during Ex Vivo Expansion.
Authors: Yoo Eun-Sun , Ahn Jee-Young , Bae Yun-Kyung , Lee Seung-Eun , Lee Sang min , Mun Yeung-Chul , Lee Kyung-Eun , Seong Chu-Myong 
Published: April 2020

Abstract
EPCs have been isolated from adult peripheral blood and bone marrow. Recently, several groups reported that two types (‘early’ & ‘late’) of EPC could be isolated from peripheral blood and bone marrow when pertinent cocktails of cytokines were used. Interestingly, early and late EPCs are different in terms of expression of surface markers, the abilities of tube formation in vitro and the capabilities of re-vascularization on hind limb ischemia models in mice. We found EPC formation during ex vivo expansion of HCB and one EPC could be found from 314 CD34+ cells from HCB based on limiting dilutional assay (ref. Stem Cells; 2003, Yoo et al). However, little is known about the characteristics of ‘early’ and ‘late’ EPCs that are derived from HCB. In this study, our aims are to isolate the ‘early’ and ‘late’ EPCs from HCB during ex-vivo HCB expansion period and to characterize the biologic properties between ‘early’ and ‘late’ EPCs. 1 x 108 mononuclear cells were plated on a 100mm culture dish coated with 50ug/ml of human fibronectin (Calbiochem) and cultured in EGM-2 BulletKit system (Clonetics). Endothelial cells were assessed by colony counts, flow cytometry, proliferation assay, RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCs were also measured by in vitro transmigration assay in the presence of VEGF and SDF-1. In results, early spindle-shaped cells (‘early’ EPCs) which were grown at first week of culture were positive for CD31, CD14 and CXCR-4. Cobblestone shaped cells (‘late’ EPC) were in peak growth at second and third weeks of culture and were also positive using above antibodies except CD14. Early EPCs had not expressed mRNA of KDR, vWF and VE-Cadherin by RT-PCR. However, late EPCs expressed high level of mRNA of those endothelial marker genes. Both early and late EPCs expressed mRNA of eNOS. Late EPC produced more nitric oxide and formed more capillary tubes than those of early spindle-shaped cells. Early EPCs were readily migrated by VEGF and SDF-1 compared with those of late EPCs. In conclusions, we have found two different types of EPCs with different biologic properties during HCB ex vivo expansion. These findings may have potential clinical applications for “cell therapy” on vascular injuries (ie, hindlimb ischemia and myocardial infacrtion). Murine models for vascular injuries are being established to test the efficacy of different types of EPCs from HCB in our Lab.
Modulation of Aldehyde Dehydrogenase and Retinoid Signaling Induces the Expansion of Human Hematopoietic Stem Cells.
Authors: Chute John P. , Muramoto Garrett G. , Whitesides John , Colvin Michael , Saffi Rachid , Chao Nelson J. , McDonnell Donald 
Published: April 2020

Abstract
Aldehyde dehydrogenases are cytosolic enzymes that convert aldehydes into carboxylic acids. Human aldehyde dehydrogenase 1 (ALDH1) is highly expressed in the liver and in hematopoietic stem cells (HSCs). Although ALDH1 is a selectable marker of HSCs, its HSC-specific function is unknown. We hypothesized that ALDH might play a critical role in HSC fate determinations since it is required for the production of retinoic acids, which are broadly implicated in tissue differentiation, tissue patterning and embryonic development in vertebrates. In this study, highly purified human CD34+CD38−lin− HSCs were cultivated with early acting cytokines, thrombopoietin, stem cell factor and Flt-3 ligand (TSF) in the presence or absence of the competitive ALDH inhibitor, diethylaminobenzaldehyde (DEAB). Treatment of human BM and CB HSCs with TSF x 7 days caused a loss of CD34+CD38− cells in culture, morphologic differentiation, amplification of committed colony forming cells (CFCs) and a loss of primitive cells capable of repopulating non-obese diabetic/severe combined immunedeficient mice (SCID-Repopulating Cells, SRCs). Conversely, 7 day culture of BM and CB HSCs with TSF plus 100 μM DEAB blocked the morphologic differentiation and lineage commitment of HSCs in culture, expanded the CD34+CD38− population, and amplified SRCs 2-fold compared to input, indicating a fundamental role for ALDH in HSC differentiation. The effects of DEAB could be reversed by the co-administration of the retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), suggesting that the ability of ALDH to produce retinoic acids is important in determining HSC fate. Via screening studies of direct ligands of RAR and RXR, we also identified a selective RXR modulator which functioned similarly to DEAB by impeding HSC differentiation and causing the 4-fold expansion of SRCs. Interestingly, treatment with either DEAB or the RXR modulator reversed the down-modulation of HOXB4 transcription that was otherwise observed in CD34+CD38−lin- cells during culture with cytokines, suggesting that inhibition of ALDH or RXR modulation may promote HSC self-renewal via discrete interactions with other regulatory pathways. Modulation of ALDH activity and retinoid signaling is a novel and effective strategy to amplify human HSCs.
Relationship between ABCG2 Expression and Side Population Stem Cell Phenotype in Murine Hematopoietic System Revealed by an Abcg2/GFP Knock-In Mouse Model.
Authors: Tadjali Mehrdad , Zhou Sheng , Rehg Jerold , Sorrentino Brian P. 
Published: April 2020

Abstract
Stem cells from a variety of tissues could be identified by a side population (SP) phenotype. We have identified an association between expression of the ABCG2 transporter and the SP phenotype, although the exact relationship between these two cellular characteristics is still not well understood. To further explore the relationship between Abcg2 expression and SP phenotype, we have generated a Abcg2/GFP reporter mouse model in which an IRES-GFP expression cassette was inserted down-stream of the stop codon of Abcg2 gene by homologous recombination, so that the expression of GFP is under the control of Abcg2 locus but endogenous Abcg2 expression was unperturbed. Immunohistochemistry of tissue sections using an anti-GFP antibody confirmed expression of GFP in tissues that normally express Abcg2, such as renal proximal tubules and intestinal epithelium. Flow cytometry analysis showed that about 10% of total bone marrow mononuclear cells expressed the Abcg2/GFP allele. Within this total GFP+ population, greater than 99% of the cells fall outside the SP region. Staining with lineage specific antibodies showed that 77% of the non-SP/GFP+ cells are Ter119+ erythroid cells. In contrast, only 0.5% of the total GFP+ population falls within the SP region. Approximately 90% of these bone marrow SP cells expressed the Abcg2/GFP allele. Transplantation studies with bone marrow cells that were depleted of lineage positive cells were then performed. All 5 mice transplanted with 100 Lin−, SP+, GFP+ cells were reconstituted in both myeloid and lymphoid lineages. In contrast, no repopulating activity was detected with up to 10,000 Lin−, non-SP, GFP− cells. When Lin−, non-SP, GFP+ cells were transplanted, only 2 of 5 animals were reconstituted with 5000 cells, and no repopulation was seen with lower cell doses. These results show a complex relationship between Abcg2 expression and the SP phenotype. Virtually all SP cells express the Abcg2/GFP allele, and these cells are highly enriched for repopulating activity in transplant assays. However, the majority of bone marrow cells that expressed the Abcg2/GFP allele were Ter119+ erythroid cells that do not bear the SP phenotype. In addition, there are non-SP, GFP+ cells that lack expression of Ter119 and other mature lineage markers. These Lin−, non-SP, GFP+ cells have relatively lower repopulating activity compared to SP cells.
Gata-Independent Regulation of Red Cell Specific Gene Expression.
Authors: Galloway Jenna L. , Wingert Rebecca A. , Thisse Christine , Thisse Bernard , Zon Leonard I. 
Published: April 2020

Abstract
During vertebrate embryonic hematopoiesis, the first blood cells can be identified by expression of the transcription factor genes scl and GATA2, followed by expression of GATA1, a gene required for the erythroid lineage. A high-throughput in situ hybridization screen in zebrafish analyzed the expression pattern of 3700 clones from a hematopoietic cDNA library and discovered 24 genes with expression in the blood. Examination of gene expression in Biklf, GATA1, GATA2, and GATA1/GATA2-deficient animals revealed that most blood genes are dependent upon GATA factors for expression rather than the Krüppel-like transcription factor Biklf. Three novel genes, expressed specifically in erythroid precursors, did not require GATA factors for their expression, demonstrating that some blood genes are regulated in a GATA-independent manner. These three genes were kelch-repeat protein (kelch repeats have been implicated in diverse cellular functions from actin binding to sequestering transcriptions factors), kiaa0650, which contains an SMC-hinge domain, and testhymin, which has no known structural motifs. By using combinations of antisense morpholinos to the known hematopoietic genes biklf , GATA1, GATA2, and scl, we were able to examine the regulation of these novel genes in double and triple knock-down embryos. While expression of kelch-repeat protein was lost in the absence of GATA1 and Biklf, expression of testhymin and kiaa0650 was maintained in GATA1/GATA2/Biklf-deficient embryos, suggesting that these similarly expressed genes are differentially regulated. As with GATA1, kiaa0650 and kelch-repeat protein required Scl for their expression in the early hematopoietic mesoderm while testhymin did not. Furthermore, loss of Scl and GATA2 did not completely ablate testhymin expression, suggesting that this gene is induced by factors upstream or parallel to Scl and GATA2. Taken together, our zebrafish studies establish a regulation of gene expression by a developmental hierarchy of specific transcription factors that act in combination during blood cell maturation.