bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Interaction between Transmembrane Domains of Glycoprotein Ibα and Ibβ Is Critical to Efficient Expression and Assembly of the Glycoprotein Ib-IX Complex.
Authors: Mo Xi , Lu Nan , Luo Shi-Zhong , Padilla Arnoldo , Lopez Jose A. , Li Renhao 
Published: April 2020

Abstract
Platelet glycoprotein (GP) Ib-IX-V complex is critically involved in the early steps of platelet adhesion and activation at sites of vessel wall injury. A functional GP Ib-IX complex comprising GP Ibα, GP Ibβ, and GP IX can be expressed in transfected cells, with efficient expression on the cell surface requiring all of its three subunits. As a first step to elucidate the 3-dimensional organizing principle of this receptor complex, we probed the interactions of its transmembrane (TM) domains. Replacement of the TM domains of either GP Ibα or GP Ibβ with poly-Leu or poly-Leu-Ala sequences markedly diminished cell surface expression of the complex, as assessed by flow cytometry and western blotting of transiently transfected CHO cells. Similar replacements of the GP IX TM domain had little effect on complex expression. TM replacements in GP Ibα or GP Ibβ also affected formation of the membrane-proximal inter-subunit disulfide bond between the two subunits. A thiol-disulfide exchange assay with the TM-containing peptides dispersed in dodecylphosphocholine micelles demonstrated a direct and specific interaction between the Ibα and Ibβ TM peptides, as the peptides preferentially formed a disulfide-linked αβ hetero-dimer rather than αα or ββ homo-dimers. Replacement mutations in the Ibα TM peptide abolished formation of the hetero-dimer. Further mutagenesis studies produced several single-site mutations within the TM domains of GP Ibα and GP Ibβ that drastically lowered the level of surface expression of the GP Ib-IX complex. These mutations also disrupted formation of the inter-subunit disulfide bond in the transfected GP Ib-IX complex as well as in the context of isolated TM peptides. These results demonstrate a specific interaction between the TM domains of GP Ibα and GP Ibβ, which can be abolished by whole-domain replacement or site-specific mutations, significantly diminishing assembly and cell-surface expression of the GP Ib-IX complex.
Ceruloplasmin Is Essential for the Mobilization of Tissue Iron Stores.
Authors: Rivera Seth , Lopez Miguel , Farshidi Dina , Gabayan Victoria , Ganz Tomas 
Published: April 2020

Abstract
In extracellular fluid, iron is in the ferric (oxidized form) but the intracellular form is ferrous iron (reduced). The outflow of iron from cells is dependent on oxidase activity that converts ferrous to ferric iron. Iron-absorbing enterocytes possess a unique iron oxidase, hephaestin. It is presumed that the circulating hephaestin paralog ceruloplasmin fulfils this role in hepatocytes and macrophages. The DiSnA mouse lacks ceruloplasmin. We hypothesized that iron homeostasis in this mouse would be unusually dependent on dietary iron because the mouse would not be able to mobilize iron from tissue stores in hepatocytes and macrophages. We fed 4-week-old DiSnA and wildtype (WT) mice a high iron (1%) diet for 4 weeks to load tissue stores. We then switched them to an iron-deficient diet and analyzed them weekly to measure iron and hemoglobin concentrations. Even on the high iron diet, DiSnA mice had lower serum iron concentrations than WT control (32.9±17.9 vs. 53.5±17.6 μM, p=0.05) but after two weeks on the iron deficient diet, the DiSnA mice had almost undetectable serum iron (4.2±1.8 μM) whereas the WT controls had only declined slightly (43.2±9.6 μM, p&lt;0.001). Iron saturation followed a similar trend. Neither WT nor DiSnA mice were anemic at baseline (Hgb = 13.9±0.3 and 13.9±1.1 g/dL, respectively; p=0.994) but by the end of two weeks, the DiSnA mice had developed anemia whereas the WT mice had not (Hgb = 10.4±0.5 vs. 12.6±0.5 g/dL; p&lt;0.001). The difference in hemoglobin concentrations persisted to the 6-week timepoint (Hgb = 8.4±0.5 vs. 12.6±1.4; p&lt;0.001). After 6-weeks on a low iron diet, iron was still present in livers and spleens of both groups. Ceruloplasmin is essential for the mobilization of iron stores to protect against iron deficiency anemia in response to periods of dietary iron deficiency.
DMT1 Mutation in a Patient with Hypochromic Microcytic Anemia: Functional Consequences and Response to Erythropoietin.
Authors: Priwitzerova Monika , Pospisilova Dagmar , Nie Guangjun , Sheftel Alex D. , Mims Martha P. , Prchal Josef T. , Divoky Vladimir , Ponka Prem 
Published: April 2020

Abstract
We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 ( DMT1 1285G&gt;C ). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full length DMT1 transcript with the patient’s point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient’s erythroid cells is severely suppressed. Because of the positive effect of erythropoietin (EPO) on the growth of patient’s BFU-Es in our in vitro studies, we have treated the patient with recombinant human EPO. The dose of 1.2 μg/kg given once a week did not lead to any improvement in hemoglobin level. However, doubling the dose of EPO (2.4 μg/kg) led to an increase in hemoglobin level from 75 to 91 g/L and to an improvement in the patient’s clinical condition.
Targeted Overexpression of the Transcription Factor XBP-1 in B Cells Promotes Plasma Cell and Lymphoplasmacytic Neoplasms in Transgenic Mice.
Authors: Carrasco Ruben D. , Sukhdeo Kumar , Protopopova Marina , German Masha , Henderson Joel , Anderson Kenneth C. , DePinho Ronald A. 
Published: April 2020

Abstract
The transcription factor X-box binding protein-1 (XBP-1) plays critical roles in the unfolded protein response (UPR), the differentiation of plasma cells, and the regulation of growth factor signaling pathways. XBP-1 is subject to regulation by alternative RNA processing producing XBP-1-spliced (S) and -unspliced (U) mRNAs encoding proteins with identical DNA-binding and bZIP domains yet distinct C-terminal transactivation domains. The weaker transactivation potential of XBP-1(U) has prompted speculation that it may influence XBP-1(S) activity as a transdominant mutant. While elevated XBP-1 expression has been reported in transformed cells, the relative ratios of these XBP-1 isoforms and associated physiological relevance in cancer are uncertain. Here, we assessed the differential impact of enforced XBP-1(S) versus XBP-1(U) transgene expression in the B cell lineage. Both transgenes elicited early onset antibody-dependent autoimmune disease characterized by elevated levels of serum immunoglobulin (Ig) and IL-6 production, increased numbers of marginal zone and mature follicular B cells in the spleen, and expanded mature B cell populations in the bone marrow. Notably, aged XBP-1(S) mice developed clonal plasma cell expansions, culminating in the human-equivalent of Monoclonal Gammopathy of Undetermined Significance (MGUS) or Multiple Myeloma (MM). Conversely, XBP-1(U) mice develop multi-organ lymphoplasmacytic infiltrates and, with advancing age, succumb to neoplasms resembling human Lymphoplasmacytic Lymphoma/Waldenstrom’s Macroglobulinemia (LPL/WM). These unanticipated genetic observations in the mouse were translated to human disease with documentation of elevated levels of XBP-1(S) in MM and XBP-1(U) in LPL/WM. Together, these results indicate that imbalances in XBP-1(S) and XBP-1(U) alters B cell lineage homeostasis and can drive two distinct types of lymphoplasmacytic neoplasms in vivo. The findings of this study, together with the known capacity of XBP-1 to regulate various cancer-relevant growth factor signaling pathways, predicts that epigenetic dysregulation of alternate XBP-1 RNA processing can promote age-associated B cell malignancies in humans.
Expression of the Iron Regulatory Peptide Hepcidin Is Reduced in Patients with Chronic Liver Disease.
Authors: Anderson Greg , Frazer David M. , Bridle Kim R. , Macdonald Graeme A. , Smith Jeffrey L. , Turlin Bruno , Subramaniam Nathan , Crawford Darrell H. , Powell Lawrie W. , Ramm Grant A. 
Published: April 2020

Abstract
Disturbances in iron metabolism often accompany liver disease in humans and hepatic iron deposition is a frequent finding. Since the peptide hepcidin, a major regulator of body iron homeostasis, is synthesised in the liver, alterations in hepcidin expression could be responsible for these effects. To investigate this possibility, we studied hepcidin expression in liver biopsies from patients with hepatitis C virus (HCV) infection, non-alcoholic fatty liver disease (NAFLD) and hemochromatosis (HC). Total RNA was extracted from the liver tissue of 24 HCV, 17 NASH and 5 HC patients, and 17 liver transplant donors (controls). The levels of mRNA for hepcidin and several other molecules involved in iron metabolism (DMT1, Dcytb, hephaestin, ferroportin, TfR1, TfR2, HFE and HJV) were examined by ribonuclease protection assay and expressed relative to the housekeeping gene GAPDH. The expression of hepcidin was significantly decreased in HCV and NASH patients relative to control liver (109±16 and 200±44 versus 325±26 respectively; P=0.008 and 0.02). We have previously reported similar findings in patients with HC, and this was confirmed in the current analysis (176±21; P=0.003). In both HCV and NAFLD patients the expression of the iron reductase Dcytb and the transferrin binding regulatory molecule TfR2 was also decreased, while the cellular iron exporter ferroportin showed a significant increase. Levels of the mRNA for the iron oxidase hephaestin were lower in HCV patients alone, while expression of the major transferrin binding molecule TfR1 was decreased only in NAFLD patients. Of particular interest was the finding that the expression of HJV (which is mutated in patients with juvenile HC) was significantly increased in NAFLD patients. No changes were seen in the expression of the iron importer DMT1 or the regulatory molecule HFE. Decreased expression of hepcidin in patients with HCV and NAFLD provides an explanation why iron homeostasis could be perturbed in these disorders. Reduced hepcidin levels would increase intestinal iron absorption and iron release from macrophages, which could contribute to hepatic iron accumulation. This in turn could lead to alterations in the expression of various proteins involved in iron transport and its regulation. Indeed most of the changes in the expression of such molecules observed in this study are consistent with this. However, the mechanisms leading to changes in the expression of hepcidin in these diseases remain to be elucidated.
Efficacy of Alpha-Lipoic Acid in Iron-Induced Oxidative Stress.
Authors: Lal Ashutosh , Suh Jung H. , Ames Bruce N. 
Published: April 2020

Abstract
Antioxidant supplementation could reduce organ damage and premature death in patients with iron overload. We evaluated the improvement in iron-induced oxidative stress with α-Lipoic acid (LA), a multifunctional thiol antioxidant and inducer of phase II enzymes, and compared it with benzylhydroxylamine (BH), a reducing agent that protects against free radicals. Human fibroblasts (IMR-90) were grown in regular cell culture medium or with ferric ammonium citrate (FAC). The intracellular iron content was measured by inductively-coupled plasma spectrometry (ICP). Either LA or BH was added on day 2, and cells grown to confluence. Oxidative stress was assessed by dichlorodihydrofluorescein diacetate fluorescence (DCF-fl). GSH:GSSG was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total intracellular iron content (mean±SEM, ng per 5x10^6 cells) increased from 66.95±6.51 in control cells to 262.26±4.69 after exposure to 25 μM FAC, and 556.26±21.15 after exposure to 125 μM FAC. The increase in iron-content of the IMR-90 fibroblasts caused significant rise in oxidant appearance at the highest concentration, as evidenced by 27.8±2.6% increase in steady-state DCF-fl compared with control cells (p &lt;.001). Addition of increasing concentrations of LA attenuated iron-mediated rise in DCF-fl in dose dependent manner. LA at concentrations above 25 μM completely abolished the iron-dependent rise in DCF-fl (91.9±2.3% for FAC+LA, p &lt;.001). BH treated cells also exhibited a dose-dependent decrease in DCF-fl, but a much higher concentration (&gt;200 μM) was needed for complete attenuation of iron-induced rise in DCF-fl (108.3±3.4%, p &lt;.001). These results suggest that LA is more effective than BH in ameliorating iron-mediated increase in oxidative stress. To further understand the basis for the efficacy of LA, intracellular GSH and GSSG were measured. Results show that LA improved the total GSH levels and its redox state in a dose dependent manner. LA and BH attenuated iron-mediated oxidant production, with LA exhibiting greater efficacy. This effect may be due to potent ability of LA to improve intracellular GSH levels and redox state. Our results suggest LA may be useful in reducing complications from iron overload.
Thiamine Deprivation Induces Reticulocytopenic Anemia in Slc19A2 Knockout Marrow: Evidence for Cell-Intrinsic Defect.
Authors: Montefusco Maria Claudia , Fleming Judith C. , Chick Gretchen , Neufeld Ellis J. 
Published: April 2020

Abstract
Megaloblastic anemia, diabetes, and sensorineural deafness characterize the autosomal recessive human disorder, Thiamine-Responsive Megaloblastic Anemia syndrome (TRMA). TRMA is due to mutations of the human SLC19A2 gene, which encodes a high-affinity thiamine membrane transporter. The murine Slc19a2 knock-out (-/-) model (Fleming et al, Mol Gen Metab; 80 (2003), 234–241) develops thiamine-dependent reticulocytopenia without megaloblastic anemia or sideroblasts, but the whole animal knockouts become ill in thiamine-depleted settings, which limit investigation of erythropoiesis. Here we investigate, by bone marrow transplantation experiments, whether the erythropoietic defect is cell-intrinsic and correlated with the correct function of Slc19a2. We conducted this study by transplanting C57BL/6J-GPIb Slc19a2−/− (−/−) or wild type (+/+) bone marrow into C57BL/6J-GPIa wild type (+/+) mice. These congenic strains were chosen to distinguish host and donor cells by GPI isozymes. After host irradiation (525 cG for 2 doses), we injected 4 X106 donor marrow cells retro-orbitally. After engraftment, confirmed by GPIa/b assay (&gt;60 days), we switched animals to either 2mg/kg chow thiamine diet (control) or 0 mg/kg thiamine (normal mouse chow is 22 mg/kg thiamine). The transplant experiments were carried in duplicate and included a total of 53 transplant recipient mice of both sexes. After 24-day diet both +/+ and −/− groups on 0 mg/kg thiamine diet lost weight (~28%). Blood parameters changed unequally. After 24 days on 0 mg/kg thiamine diet mice transplanted with −/− bone marrow reveled a significant increment of reticulocyte mean cell volume (MCV) (23% with P&lt; 0.01) together with a relevant decrease of red blood cell count (46% and P&lt; 0.01). A severe leukopenia diet correlated was also observed in both groups of mice on thiamine-depleted diet. The blood parameters of mice on 2-mg/kg diet did not reveal significant changes along the entire diet period. These results confirm the involvement of Slc19a2 membrane transporter in the impaired thiamine cell uptake and allow us to say that the erythropoietic defect we first observed in Slc19A2 KO mice is not only diet related, but due to a cell-intrinsic marrow defect.
Tables. Peripheral blood parameters of bone marrow transplanted mice at baseline and after 24 days on depleted thiamine diet.
BASELINE Donor WBC RBC MCV Retic −/− mean (StDev) 20.41 (4.23) 10.5 (3.46) 61.83 (2.38) +/+ mean (StDev) 20.63 (2.36) 9.24 (1.45) 67.4 (3.03) P 0.9140 0.4257 0.0136 24 days on 0 mg/kg thiamine diet Donor WBC RBC MCV retic St Dev = Standard Deviation P = probability −/− mean (StDev) 8.53 (1.75) 6.79 (1.65) 76.18 (1.24) +/+ mean (StDev) 6.51 (3.16) 6.09 (1.37) 65.23 (5.04) P 0.3232 0.0321 0.0011
Differential Patterns of Transcriptional Protein Expression May Explain Functional Differences between Hematopoietic Progenitors Derived from Human ESC’s and Fetal Hematopoietic Tissues.
Authors: Shaaban Aimen F. , Golos Thaddeus G. , Thomson James A. , Rajesh Deepika 
Published: April 2020

Abstract
Recent studies suggest that development of human embryoid body-derived hematopoietic progenitor cells (EB-HPC’s) mirrors that of primitive yolk sac-derived hematopoietic progenitors (YS-HPC’s) in their expression of transcription factors known to be critically associated with hematopoietic development. However, the findings of these studies were limited to molecular characterization of heterogeneous EB cultures rather than analysis of hematopoietic lineage-committed cell subsets. We wondered: 1) if the expression patterns were consistent at the protein level in both bulk EB cultures and subsets of hematopoietic-lineage committed cells; and 2) if these expression patterns differed from those found in definitive HPC’s harvested from early fetal hematopoietic tissues. To answer these questions, we compared the intracellular expression of critical transcriptional regulatory proteins from human and rhesus EB-derived HPC’s (hEB-HPC’s and rEB-HPC’s) with hematopoietic progenitors from rhesus fetal liver (rFL-HPC’s) and rhesus placental blood HPC’s (rPB-HPC’s). Rhesus macaque fetal tissue was used in lieu of scarce human fetal tissues. Using multi-color intracellular flow cytometry we correlated the expression of transcriptional proteins (SCL/TAL1, GATA-1, GATA-2, Oct-3/4, HOXB4) with cell surface determinants of hematopoietic commitment (CD34, CD31, CD45) in hEB-HPC’s, rEB-HPC’s, rFL-HPC’s and rPB-HPC’s. Human and rhesus EB16 cells were harvested from identical culture conditions and compared to 0.34 gestation rFL-HPC’s and rPB-HPC’s. The frequency of CD45+ cells was higher in hEB-HPC’s (31%) and rPB-HPC’s (46%) compared to rFL-HPC’s and rEB-HPC’s. A much higher frequency of GATA-1 and HOXB4 expressing cells was seen in both of the fetal cell types when compared to either rhesus or human EB’s. Additionally, the frequency of GATA-2 and SCL expressing cells was comparable in all cell types. Lastly, the expression of Oct3/4 was higher in rPB-HPC’s and rFL-HPC’s when compared to bulk EB cultures. However, analysis of subsets of EB cells expressing hematopoietic antigens (CD45, CD34) revealed co-expression of Oct-3/4 on both CD45+ and CD34+ cell fractions. From these findings, we conclude:
critical differences exist in GATA-1 and HOXB4 expression between HPC’s derived from EB cultures and HPC’s harvested during early definitive fetal hematopoietic development; and human EB-derived hematopoietic progenitor cells express persistently high levels of Oct-3/4.
The differences in the expression of these critical transcriptional factors at the protein level may explain the observed functional differences between EB-derived HPC’s and definitive HPC’s from fetal and adult sources.
Slug Antagonizes p53-Mediated Apoptosis of Hematopoietic Progenitors by Repressing Puma.
Authors: Wu Wen-shu , Heinrichs Stefan , Xu Dong , Adams Jerry M. , Look A. Thomas 
Published: April 2020

Abstract
The discovery that Slug has a vital anti-apoptotic role in the normal response of hematopoietic progenitors to genotoxic stress compelled us to investigate the underlying cellular and molecular mechanism(s) of this effect. Here we show that, although the development of myeloid progenitors in Slug−/− mice is not impaired under steady-state conditions, their ability to repopulate the compartment after γ-irradiation is reduced. We demonstrate that the radiation-induced death of Slug−/− mice exclusively reflects bone marrow failure and show that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. We also establish that Slug confers radioprotection by inhibiting the mitochondria-dependent apoptotic pathway activated by γ-irradiation in hematopoietic progenitors. We show that Slug acts as a transcriptional repressor by directly antagonizing p53-mediated upregulation of the BH3-only gene Puma, which was recently shown to encode a critical mediator of p53-induced apoptosis. Finally, we provide evidence for a novel feedback loop in the hematopoietic progenitor cells after DNA damage: Slug is itself induced by p53. Thus, the survival of hematopoietic progenitor cells after genotoxic stress relies on induction of Slug by p53 and Slug-mediated repression of Puma.
A Natural Variant Sequence in the AHSP Gene May Impact Severity of Beta-Thalassemia.
Authors: dos Santos Camila O. , Zhou Suiping , Albuquerque Dulcineia M. , Saad Sara , Weiss Mitchell J. , Costa Fernando F. 
Published: April 2020

Abstract
Alpha-hemoglobin stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds the alpha chains of hemoglobin, preventing their precipitation and deleterious effects. Loss of AHSP exacerbates alpha globin precipitation and anemia in a murine model for beta thalassemia. In vitro, recombinant AHSP inhibits the production of reactive oxygen species (ROS) by alpha hemoglobin. To further define the role of AHSP as a modifier of beta-thalassemia, we analyzed AHSP sequences for mutations in a large population of beta-thalassemic and control subjects. A single nucleotide change, from A to T at position 12888 (Genebank accession number AC106.730.2) in the third exon of the AHSP gene was identified, converting asparagine 75 to leucine (N75I). We detected a patient who was heterozygous for both beta-thalassemia (b39/bA) and the AHSP-N75I mutation. She presented with an unusually severe anemia that required regular blood transfusion. The father is hematologically normal, without any thalassemia mutation and heterozygous for AHSP-N751. The mother is heterozygous for the beta 39 mutation with classical mild hypochromic microcytic anemia and no mutation in the AHSP gene. Of 282 unrelated control subjects tested, one (0.35%) contained the N75I mutation. Analysis of red blood cells from this subject revealed normal hemoglobin indices but a small number of Heinz bodies, suggesting hemoglobin precipitation. This finding is similar to what we observed previously in mice that are heterozygous for an AHSP null allele. To investigate how the N75I mutation affects AHSP function, we studied the biochemical properties of the recombinant mutant protein. Surface plasmon resonance showed that the binding affinity of AHSP N75I for alpha hemoglobin is normal. Importantly, compared to wild type AHSP, the N75I mutant protein exhibited significantly reduced capacity to inhibit ROS production by alpha hemoglobin. Hence, AHSP N75I may be less effective at conferring protection from oxidative-mediated damage by free alpha hemoglobin in erythrocytes. These effects, when coupled with beta thalassemia, could result in more severe anemia, implicating AHSP N75I as a potential genetic modifier.