bluuebunny/crossref_metadata_2025 huggingface dataset:
Computational Design of a Model for the Platelet Integrin αIIbβ3.
Authors: Metcalf Douglas G. , DeGrado William F. , Bennett Joel S.
Published: October 2019

Integrins, the principal receptors linking the cytoskeleton to extracellular ligands, are responsible, in large part, for platelet adhesion and aggregation. Like many membrane spanning proteins, the molecular structure of integrins has been difficult to determine, although crystal and NMR structures are available for water soluble portions of αVβ3 and αIIbβ3. In addition, models for the αIIb and β3 transmembrane (TM) heterodimer have been obtained using computational methods, but with conflicting results. Accordingly, we employed two different modeling strategies to distinguish between existing models. First, we used a Monte Carlo-simulated annealing (MCSA) protocol that features a selective advantage for structures that are consistent with previously characterized αIIb and β3 TM domain point mutations. During each step of the protocol, we calculated the effects of selected mutations and assigned a penalty to the step if it produced a structure that was inconsistent with experimental results. Second, we threaded the αIIb and β3 TM sequences onto a database of membrane helix dimers and rank-ordered the structures based on their calculated energies and their agreement with the cysteine crosslinking experiments reported by Luo et al (PLoS Biol 2:776–86, 2004). The MCSA and threaded TM models were similar with a root mean squared deviation (RMSD) of 1.5 Å. Moreover, they differed from a model reported by Kay Gottschalk (Structure 13:703–12, 2005) most notably by an ~50° rotation of the αIIb TM helix about its helical axis. This positions αIIb residue Leu980 in the TM interface and in van der Waals contact with β3 residue Gly708, a result consistent with mutagenesis experiments. Further, the αIIb and β3 TM helices pack along conserved glycine residues (Gly972 and Gly976) in the GXXXG motif of αIIb with a right-handed crossing angle of −18°. To create a model for the full-length integrin, the N-termini of the TM model were connected to the crystal structure of the αVβ3 extracellular domain and its C-termini were connected to the corresponding NMR structures of the αIIb and β3 cytosolic domains. The model predicts an αIIb Arg995-β3 Asp723 salt bridge in the membrane proximal cytosolic domains and a change in the secondary structure of the extracellular domains from an extended to a helical conformation that occurs at Pro965 in αIIb and Pro693 in β3, just proximal to the TM domains. Finally, the full length model provides insight into distal interactions since its extracellular portion is held close to the membrane and is therefore sterically inaccessible to large ligands such as fibrinogen, whereas cytosolic regulatory sites such as the talin binding site in β3 are accessible and not sequestered by α/β interactions. In conclusion, we have modeled the αIIbβ3 TM heterodimer using two fundamentally different computational strategies, both of which converge upon similar structures that were used to construct a model for the full-length integrin.
The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.
Authors: Mo Xi , Lopez Jose A. , Li Renhao
Published: October 2019

Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.
Hepcidin Suppression Following Phlebotomy Is Regulated by an Erythropoietic Regulator Found in Serum.
Authors: Rivera Seth , Pak Mihwa , Lopez Miguel A. , Gabayan Victoria , Ganz Tomas
Published: October 2019

Increasing erythropoiesis requires a dramatic increase in iron absorption or the release of iron from tissue stores. Since the iron regulatory hormone hepcidin blocks both absorption and release from stores, it is suppressed during erythropoiesis. We have recently shown that hepcidin is primarily suppressed by an erythropoietic regulator rather then anemia, hypoxia, or erythropoietin (Pak et al, Blood, epub/in press). To further understand the regulation of hepcidin during erythropoiesis, we subjected groups of eight mice to phlebotomy and sacrificed them at various time points over 3 weeks. Hepcidin mRNA fell 25-fold by day 2 (p<0.001) and did not begin to rise until day 9. Hemoglobin rose well before hepcidin mRNA began to increase confirming that anemia was not the erythropoietic regulator. Serum iron never fell, excluding this as the erythropoietic regulator. Erythropoietin levels spiked early but returned to normal well before hepcidin. Reticulocyte counts increased shortly after the fall in hepcidin and remained elevated after hepcidin mRNA returned to normal. We therefore postulate that the erythropoietic iron regulator is either made by or in concert with early erythroid progenitor cells. Using primary hepatocytes treated with serum from erythropoietin stimulated or control mice, we also demonstrate that the erythropoietic regulator of hepcidin expression is present in the circulation. Experiments are underway to isolate the erythropoietic regulator of hepcidin from serum.
Rapamycin Analogs Reduce mTORC2 Signaling and Inhibit AKT Activation in AML.
Authors: Zeng Zhihong , Sarbassov Dos D. , Giles Francis J. , Samudio Ismael , Yee Karen W.L. , Jackson C. Ellen , Sabatini David M. , Andreeff Michael , Konopleva Marina
Published: October 2019

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin-insensitive, and was recently shown to regulate the pro-survival kinase AKT by phosphorylation on Ser473 (Sarbassov Science 2005;307 and Mol Cell 2006;22). We investigated the molecular effects of mTOR inhibition by rapamycin analog CCI-779 in AML cells. Unexpectedly, CCI-779 not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased phosphorylation of p70S6K, 4EPB1 and reduction in Glut-1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-Cyclins in AML cell line and 5 of 8 primary AML samples in vitro. Similar observations were made in samples from patients with hematological malignancies who were treated with the rapamycin analogs temsirolimus or everolimus: the levels of Ser473 phosphorylated AKT decreased in 3/5 patient samples at 1 or 24 hour(s) of temsirolimus treatment, and in 6/8 patient samples treated with everolimus. In the 9 samples in which AKT was inhibited, ≥2-fold decrease in Cyclin D1 mRNA was observed in 5, Cyclin D2 in 3, both, Cyclin D1 and D2 in 1 sample, and Glut-1 in 4 patient samples. In 7 of the 9 patients in whom AKT was inhibited, a >50% decrease in peripheral blood absolute blast count (3 AML, 1 ALL) or absolute lymphocyte count (1 CLL) for >1 week duration was documented, and two patients with RAEB-1 had improvements in platelet counts, one fulfilling the criteria for clinical response (hematological improvement). No change in peripheral blood counts or progression of leukemia was seen in 6 patients. Of these, decrease in pAKT was observed in 2, no change in 3 and increase in 1 (Fisher exact two-tailed p= 0.021). Altogether, our study provides first evidence that rapamycin analogs inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias.
Formation of Nitroso Compounds in the Reaction of Nitrite and Hemoglobin.
Authors: Basu Swati , Huang Jinming , Huang Zhi , Jeffers Anne , Jiang Alice , He Xiaojun , Azarov Ivan , Seibert Ryan , Patel Rakesh P. , Hogg Neil , King Bruce , Gladwin Mark T. , Kim-Shapiro Daniel
Published: October 2019

Nitrite is being studied as a therapeutic agent for several pathologies, largely due to the demonstration that infusions of nitrite into the human forearm results in increased blood flow. Thus, nitrite may serve as a storage pool for the endothelium derived vasodilator, nitric oxide (NO). Nitrite is known to react with deoxyhemoglobin to form NO, which would need to diffuse from the red blood cells to the smooth muscle cells in order to activate its target enzyme soluble guanylyl cyclase. However, hemoglobin scavenges NO extremely quickly so that NO formed in the red blood cell cannot get out. The export of nitrite-derived NO activity is likely to require the formation of either a diffusible intermediate or alternative species for the transport of NO. One of the suggested intermediates is S-nitrosohemoglobin, small amounts of which have been reported to have formed during nitrite infusion studies. Other investigators recently reported that S-nitrosohemoglobin is directly formed in the reaction of nitrite and hemoglobin via the formation of a quasi-stable Fe(III)NO-hemoglobin intermediate. We have used absorption spectroscopy, electron paramagnetic resonance spectroscopy, and several chemiluminescence-based assays to the nitrite/hemoglobin reaction, focusing on the formation of S-nitrosohemoglobin and other nitroso species such as S-nitrosoglutathione (GSNO). We have also developed a sensitive chemiluminescence-based assay to explore Fe(III)NO-Hb formation. We applied these techniques to a variety of conditions, varying oxygen saturation and reactant concentrations in cell-free and red cell encapsulated hemoglobin. Based on our results, we conclude (contrary to several other recent reports) that little or no nitroso species are formed in the reaction of nitrite and hemoglobin at neutral pH (7.4) and a quasi-stable Fe(III)NO-Hb intermediate is also not formed. We find that, under thoroughly deoxygenated conditions, one nitrite molecule results in the formation of one Fe(II)NO-Hb and one MetHb species, with some nitrite bound MetHb being formed subsequently. Importantly, we find that at pH 6.5, significant nitrosation does occur. These data suggest that much or all of the nitroso species measured in nitrite therapeutics may be formed in metabolically active muscle or other tissues of mild acidity.
Lysophospholipid Acylation in RBC.
Authors: Soupene Eric , Kuypers Frans A.
Published: October 2019

The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). We recently identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC. None of the family members of LPLAT have been identified in RBC to date. LPC, either generated in the RBC or taken up from plasma, is rapidly acylated by RBC suggesting an important role for Lysohosphatidylcholine-acyl transferase (LPCAT) in RBC. We report the identification and characterization of LPCAT, the enzyme that generates PC from LPC and acylCoA. We identified the RNA expression of LPCAT, an approximately 60kD protein, in reticulocytes, confirming proteomic studies suggesting the presence of this protein in adult RBC membranes.). It is a modular protein containing an acyltransferase domain at the amino-terminus, three predicted membrane spanning domains, and a putative calcium binding site at the C-terminus, distinguishing it from the lysophosphatidic acid acyltransferease (LPAAT). The putative LPCAT was expressed in E. coli. It was found in the E. coli membrane fraction, and was able to use oleoyl-CoA and LPC as substrates to generate PC. Lysophosphatidic acid (LPA) was not acylated by this protein. In contrast the previously identified LPAAT (1) expressed in E. coli, utilized LPA but not LPC, indicating LPL specificity of these enzymes. Radioactive fatty acid added to RBC is also incorporated in phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). Sequence analysis suggests that two other proteins present in the genome of mammals are homologues of LPCAT. We hypothesize that these putative acyltransferases are responsible for the acylation of lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl serine (LPS). These proteins are essential to maintain a proper glycerophospholipid composition of the RBC membrane and thereby viability of the cells. A dysfunction of this system may underlie the observed differences in phospholipid molecular species composition in subpopulations of sickle cells contributing to sickle cell pathology. A complete description of these proteins involved in the maintenance of glycerophospholipid composition of RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.
Recessive Congenital Methaemoglobinaemia: Three New Mutations in the NADH-Cytochrome b5 Reductase Gene.
Authors: Fermo Elisa , Bianchi Paola , Vercellati Cristina , Marcello Anna P. , Barcellini Wilma , Marangoni Ornella , Garatti Massimo , Zanella Alberto
Published: October 2019

Recessive congenital methemoglobinemia (RCM) is caused by deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (cytb5r). The cytb5r gene (DIA1) is localized on chromosome 22q13-qter and contains nine exons; tissue-specific alternative transcripts originate 275-amino acid soluble and 300-amino acid membrane-bound isoforms. Cytb5r deficiency exists in two distinct clinical forms: in Type-I the enzyme deficiency is restricted to the red cell-soluble cytb5r isoform and results in cyanosis; in Type-II the enzyme defect involves both the soluble and membrane-bound isoforms, and severe neurological impairment is also present. More than 40 different mutations of DIA1 gene have been reported in RCM: disruptive mutations (stop, frameshift, splicing) are in general associated with the severe Type-II disease. We report the clinical, hematological and molecular characterisation 6 new patients affected by RCM (Table 1). Mutations reported in italic are new. The study of cytb5r enzyme showed a drastic reduction of activity in all patients. Parents displayed intermediate values characteristics of an heterozygous state. Mutation IVS5+2 t-c at the homozygous state was found in Type-II RCM (case 1) as already reported by Yilmaz et al (2005) in one patient; this variant is likely to cause alteration of mRNA splicing and subsequent absence of functional protein in all tissues, therefore accounting for the severe clinical pattern. V252M is the most common DIA1 gene mutation and has been reported in both Type-I and -II homozygous patients; the finding that this mutation in association with the new, very disruptive Gln28STOP mutation results in a benign phenotype (case 4), suggests that V252M is more likely to behave as a “Type-I” mutation. In case 6, in spite of cyanosis present since childhood, diagnosis of RCM Type-I was made in adult age because of polycythemia. In fact, significant compensatory elevation of hemoglobin concentration is sometimes observed in patients with congenital methemoglobinemia.

Patient, (age) Type MetHb(%) Hb (g/dL) Hct (%) Cytb5r activity (IU/gHb) Mutations Effect 1 (8months) II 12.1 12.2 36.5 1.4 IVS5+2 t-c/IVS5+2 t-c Splicing/splicing 2 (2months) I 26.2 11.9 35.6 0.9 Cod.143 GGC-GAC/Cod.143 GGC-GAC Gly143Asp/Gly143Asp 3 (4months) I 12.4 16.6 49 1.7 Cod 218 CGA-TGA/Cod 105 GTG-ATG Arg218STOP/Val105Met 4 (1month) I 25.7 14.9 45 0 Cod 28 CAG-TAG/Cod. 252 GTG-ATG Gln28STOP/Val252Met 5 (1month) I Nd 12.2 35 1.9 Cod 46 CGG-TGG/Cod. 252 GTG-ATG Arg46Trp/Val252Met 6 (47years) I 23.7 18.6 55 0 cod 144 CCC-TCC/cod 252 GTG-ATG Pro144Ser/Val 252Met 0.5–1.5 12–16 39–49 15.4–23.1
Consumption of C.E.R.A. and Epoetin Beta in a Cellular Assay: UT-7 Consumption Model.
Authors: Jarsch M. , Brandt M. , Haselbeck A.
Published: October 2019

Continuous Erythropoietin Receptor Activator (C.E.R.A.), an innovative erythropoietic agent with unique receptor activity, is currently in development to provide correction of anemia and stable maintenance of hemoglobin (Hb) levels at extended administration intervals up to once monthly in patients with all stages of chronic kidney disease, and is also in development for the treatment of chemotherapy-induced anemia. C.E.R.A. has an equivalent half-life in humans of approximately 130 h following subcutaneous or intravenous administration. Clearance of erythropoietic agents may occur by binding, internalization and degradation in receptor bearing cells in the bone marrow. A functional assay was developed to evaluate the consumption of C.E.R.A. and epoetin beta in a UT-7 cellular system (a human acute myeloid leukemia cell line whose growth is dependent on the presence erythropoietic and other growth factors). UT-7 cells were incubated in the presence of C.E.R.A. (1000 pM) or epoetin beta (100 pM) for 72, 96 or 120 h. The cell supernatants were then harvested and the amounts of remaining growth factor were determined by a validated erythropoietin ELISA. As controls, the growth factors were incubated for the same times in medium only. Five independent identical experiments were conducted for each compound. Mean percentages of C.E.R.A. and epoetin beta were calculated relative to the control values (set at 100%). C.E.R.A. 1000 pM and epoetin beta 100 pM were shown to be equipotent, stimulating the proliferation of UT-7 cells to the same degree. There was a highly significant difference (P=1.9×10−9) between C.E.R.A. and epoetin beta for the time course of concentrations relative to controls. For epoetin beta 100 pM, the relative concentration decreased significantly over time, while for C.E.R.A. 1000 pM no statistically significant changes were observed. Thus, C.E.R.A. was still present in the culture supernatant at close to 100% after 4–5 days’ incubation, while the epoetin beta concentration had declined to approximately 20%. In conclusion, at the end of the incubation time the C.E.R.A. concentration was still sufficient to maintain the initial level of cell stimulation. However, at the end of the 96 h incubation the epoetin beta concentration dropped to a level that was insufficient to produce further stimulation. The UT-7 consumption model thus emulates the in vivo situation in humans with regard to receptor mediated elimination of the compounds and stimulation of erythroid progenitors.

This study provides further evidence that C.E.R.A. interacts differently with the erythropoietin receptor relative to epoetin beta. Preclinical studies have shown that these properties translate into more continuous stimulation of erythropoiesis in vivo compared with epoetin beta and Phase III data indicate that C.E.R.A. allows predictable Hb maintenance in direct conversion from more frequently dosed agents.
A New Generation of γGlobin Gene Inducers Act on Novel Molecular and Cellular Targets.
Authors: Perrine Susan , Trudel Marie , Castaneda Serguei A. , Mankidy Rishikesh , Boosalis Michael S. , Eiymo Marthe-Sandrine , White Gary L. , Bohacek Regine , Faller Douglas V.
Published: October 2019

High-potency, non-cytotoxic oral therapeutics to stimulate both fetal globin expression and erythropoiesis are needed for optimal definitive therapy of the beta-hemoglobinopathies and thalassemias. Butyrate and phenylbutyrate have been the most active of the non-chemotherapy inducing agents, but their efficacy is limited by anti-proliferative effects. A discovery program to identify more potent fetal-globin inducing compounds has been pursued utilizing pharmacophore and pseudoreceptor models to screen chemical libraries for additional inducing agents, based initially on short chain fatty acid derivatives. From a library of 10,000 compounds, our pseudoreceptor model predicted that 30 candidate compounds would bind a structural receptor which results in induction of the γ globin gene promoter. Of the 30 candidates, two compounds (RB 4 and 9) were found to have inducing activity comparable to butyrate in reporter gene assays (producing 2-fold induction), two candidates (RB7 and 29) had higher potency (3–4 fold induction), and 9 compounds had intermediate (lower) inducing activity. Second, five compounds identified through the molecular modeling stimulated proliferation of cord blood erythroid progenitors significantly, even in the absence of any added cytokines. Four of the novel compounds induced fetal globin mRNA (assayed by RT-PCR) in erythroid progenitors by 1.5 to 7-fold over control cells, at concentrations one log lower than previously identified inducers, while butyrate induced γ mRNA by 2-fold but reduced erythroid colony growth by 15%. Of compounds studied in vivo, three candidate compounds induced γ globin mRNA (by 2 to 6-fold) over basal levels in baboons, or by 2–15-fold in mice transgenic for the human γ globin genes. Molecular mechanisms of fetal globin induction by the high-potency compound RB7 resulted from displacement of an HDAC3/NCoR repressor complex from the γ globin promoter, with concurrent local histone acetylation, binding of a remodeling complex, and recruitment of RNA Pol II. These studies together identify 1) novel therapeutic targets, and 2) therapeutic candidates with multiple beneficial actions for treating beta globin disorders and other anemias. Development of a high-potency, orally tolerable therapeutic to benefit even severely anemic thalassemia patients should now be feasible.
A Novel beta-delta Globin Gene Fusion Leads to Over-Expression of Delta Globin Chain and a Mild Thalassemia Intermedia Phenotype When Co-Inherited with β0-Thalassemia.
Authors: So Chi-Chiu , Chan Amy Y.Y. , Tsang Stella T.Y. , Lee Anselm C.W. , Au Wing-Yan , ShiuKwan Edmond , Chan Li-Chong
Published: October 2019

Anti-Lepore hemoglobins are rare βδ fusion variants that arise from nonhomologous crossover during meiosis, resulting in a δ-βδ-β configuration in the β globin cluster on the short arm of chromosome 11. No such variant has previously been reported in Chinese. We studied 2 unrelated Chinese families with anti-Lepore due to a novel βδ fusion. A moderate increase of Hb A2 to between 16.8 and 19.2% of total Hb was seen in 4 clinically and hematologically normal carriers, while in 1 patient with concurrent β thalassemia allele and mild thalassemia intermedia phenotype, the Hb A2 was markedly raised to 42.2%. Potential βδ fusion gene was amplified using PCR with primer 5′ to β globin gene Cap site and 3′ to δ globin gene poly A region in these 5 subjects. Direct sequencing of the PCR products revealed a crossover between the β and δ globin gene within a 54 bp region spanning the junction of CAP and exon 1 in all 5 subjects. Active transcription of this novel βδ fusion gene was studied in 2 subjects (1 normal carrier and the patient with thalassemia intermedia). Using primer pair 5′ to β globin gene Cap site and at the 3′ end of δ globin gene exon 3, a 655 bp PCR product was obtained from cDNA in both subjects. Direct sequencing confirmed the presence of a βδ fusion mRNA. The predicted protein product of this fusion gene was identical to δ globin, since only the 5′ untranslated region was β specific. Determination of α/β mRNA ratios by quantitative real-time PCR was performed in 2 normal carriers (ratio 3.28 and 3.95) and the patient with thalassemia intermedia (ratio 25.59). These were compared with ratios ± standard deviation from 20 normal controls (1.92±0.55), 5 unrelated patients with β thalassaemia minor (17.23±5.38) and 3 unrelated patients with β thalassemia major (139.21±39.50). Mild down-regulation of the β gene in cis due to this interposed βδ fusion gene was demonstrated. Although heterozygotes of this novel anti-Lepore Hb had normal red cell indices and were clinically silent, compound heterozygotes with β0 mutation in trans produced a mild thalassemia intermedia phenotype with a markedly raised Hb A2 level that might mimic clinically mild Hb E-β+ thalassemia. Awareness of this new anti-Lepore Hb will help to resolve diagnostic problems in regions with significant prevalence of globin disorders.
Haplotype Analysis of -α3.7 Chromosomes in Israeli Ethnic Groups Reveals Unexpected Heterogeneity and Demonstrates Ashkenazi Founder Groups in Carriers of α-Thalassemia.
Authors: Rund Deborah G. , Shaulov Adir , Filon Dvora
Published: October 2019

α-thalassemia (α-thal) is among the world’s most common single gene disorders, whose prevalence in the “malaria belt” is attributed to a selective advantage of carriers. Our previous studies demonstrated a high frequency of deletional α-thal (nearly all heterozygotes or homozygotes for -α3.7) in Ashkenazi Jews (carrier frequency of 7.9%, allele frequency of 0.04) (Rund et al, 2004). Ashkenazim resided in temperate climates for centuries and were not subject to malarial selection pressure, and their carriership for β-thalassemia is very low (estimated <0.1%). To elucidate the genetic mechanism(s) responsible for this high frequency of α-thal, we performed α-globin haplotype analysis on 170 chromosomes (chromos) of 85 homozygotes for -α3.7. We compared chromos of several ethnic groups: Jews (Ashkenazim: 54 chromos, Yemenites: 54 chromos, Iraqis: 14 chromos, others: 14 chromos), Arabs (28 chromos), and Druze (6 chromos). Using PCR and digestion with ApaI and RsaI, it was determined that all but three of the chromos are of the -α3.7I type. Haplotype analysis was performed for polymorphic sites identified by Higgs (1986), using PCR and restriction enzyme digestion. Altogether, 13 haplotypes were found. Unexpectedly, at least 5 haplotypes were found among the Ashkenazim with a large number of chromos carrying unknown haplotypes. Interestingly, 26/54 of Ashkenazi chromos carried haplotype IIIb which is found rarely in Europe and Saudi Arabia but most commonly in Melanesia and Papua New Guinea (Flint, 1992). In contrast, only 3/116 nonAshkenazi chromos carried haplotype IIIb. Interestingly there was little overlap in haplotypes between Ashkenazim and the various ethnic groups studied including the other Jewish groups, with 2 exceptions. First, Arabs and Yemenite Jews each were found to have around 50% chromos which carried haplotype Ia. Additionally, 10% of Ashkenazim and 20% of Yemenites had chromos carrying haplotype IIh, which is a haplotype originally described in an Australiam Aboriginal tribe (Roberts-Thomson, 1996). There was no overlap between Arabs and Druze. In conclusion, α-globin haplotype analysis demonstrates diversity within an apparently homogeneous ethnic group (Ashkenazim homozygous for -α3.7) and demonstrates founder effects in Ashkenazim carrying α-globin gene rearrangements.
The Physiology of Prothrombin Gene Expression Integrates RNA Polyadenylation and Splicing in a Novel Regulatable 3′ RNP-Complex.
Authors: Danckwardt Sven , Gentzel Marc , Gehring Niels H. , Kaufmann Isabelle , Neu-Yilik Gabriele , Wilm Matthias , Hentze Matthias W. , Kulozik Andreas E.
Published: October 2019

The functional analysis of the common prothrombin (F2) 20210*A allele has recently revealed gain-of-function of 3′end processing as a novel genetic mechanism predisposing to human disease. The general susceptibility of the F2 mRNA for gain-of-function is further exemplified by F2 20209*T and F2 20221*T, and can be explained by an unusual architecture of non-canonical 3′end formation sequence elements: Specifically, the F2 3′ untranslated region (3′UTR) contains a stimulatory upstream sequence element (USE) that compensates for the weak functional activities of the cleavage site and the downstream U-rich element in the F2 3′ flanking sequence.

We now show that the F2 USE promotes 3′end formation in a position- and sequence-dependent manner, stimulating the step of mRNA polyadenylation rather than cleavage, and identify specific proteins that interact with the USE. Unexpectedly, the USE RNP includes splicing factors, components of the 3′end processing machinery and AU-rich sequence element-binding proteins (ARE-BP). We demonstrate that the splicing factors U2AF35 and U2AF65, hnRNPI/PTB, PSF/SFPQ and p54nrb/NonO promote 3′end formation via the USE contained in the 3′UTR uncovering a novel and more general functional link between these splicing factors and mRNA 3′ end formation. We propose a model of USE-directed 3′ end processing that involves a novel mRNP that integrates different nuclear pre-mRNA processing steps. Furthermore, the involvement of ARE-BP in this mRNP reveals an intriguing potential for a post-transcriptional regulation of prothrombin gene expression through external stimuli. Our data thus implicate USE-dependent RNP-complex formation in the regulated physiology of prothrombin gene expression specifically and in hemostasis (and other thrombin-dependent processes) more generally.
Function of Beta2-GPI in Inhibiting the Oxidized Phospholipids Dependent APC Activity by Antiphospholipid IgG from APS Patients.
Authors: Hu Yanqing , Esmon Charles T. , Esmon Naomi L.
Published: October 2019

Beta2-GPI (ß2GPI), a plasma glycoprotein with phospholipid-binding properties, is the actual target antigen for autoimmune type antiphospholipid antibodies. Certain groups of these antibodies exert lupus anticoagulant (LA) activity by directly inhibiting the Protein C anticoagulant pathway and associated with an increased risk of thrombotic disease. Our previous studies have shown that the activated protein C (APC) activity and the ability to be inhibited by antiphospholipid antibodies associated with thrombosis are strongly augmented by the presence of Phosphatidyl-ethanolamine (PE) and phospholipid oxidation. In this study, we investigated the relationship between ß2GPI and anti-APC activity by IgG derived from APS patients.

IgG from patients with APS syndrome were tested using a modified oxidized PE containing phospholipids dependent dRVVT test, in the presence of APC at concentration of 0.2ug/ml, the clotting times was prolonged to 3 fold of the baseline level, when the anti-phospholipids IgG antibodies added into the system, its showed a significant impact on the APC prolongation clotting times in the presence of ß2GPI. However, no affect was observed on the APC prolongation clotting times in the absence of ß2GPI. To eliminate the possible confounding effect from other plasma component, we investigated the effect on factor Va inactivation by APC in a purified system. As expected, the inhibition was observed only when the B2GPI was present. We also found that the presence of beta2GPI-IgG complex had its strongest effect when Protein S was included.

We conclude that ß2GPI is necessary to inhibit the oxidized phospholipids dependent APC activity by IgG antibodies from APS patients. On oxidized lipids surface, ß2GPI -APS IgG complex had its strongest effect on APC activity. This is a potentially important result indicating the functional assay may be more sensitive for pathological antibodies.
Evidence for an Enhanced Fibrinolytic Capacity in Liver Cirrhosis Measured with a New Global Fibrinolysis Test in Whole Blood.
Authors: Leebeek Frank W.G. , Kock Edith L. , Guimaraes Ana H. , Murad Sarwa D. , Janssen Harry L. , Rijken Dingemans C.
Published: October 2019

It is known that liver cirrhosis is associated with hyperfibrinolysis, which might contribute to bleeding problems of patients with this disease. However, hyperfibrinolysis has been assessed by using tests (such as euglobulin, plasma or diluted whole blood clot lysis assays), which do not reflect all changes that might occur in the fibrinolytic parameters in blood. A recent study even questioned the presence of a hyperfibrinolytic state in nonbleeding cirrhotic patients, since no evidence of increased fibrinolysis was observed with a new plasma-based clot lysis assay (Gastroenterology2001;121:131). Therefore we decided to re-investigate the fibrinolytic state of cirrhotic patients with a recently developed test for global fibrinolytic capacity (GFC) using undiluted whole blood. Non-anticoagulated blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The GFC was determined immediately after collection by clotting the blood, incubating the clots during 3 hours at 37 degrees and then measuring the generated fibrin degradation products. Various haemostatic parameters were determined in plasma samples by using functional activity assays. The median GFC (25th–75th percentile) increased from 1.7 (0.8–3.5) mg/l in the controls to 4.0 (1.6–13.0) mg/l in Child-Pugh A (n.s.), to 11.1 (2.3–31.9) mg/l in Child-Pugh B (P<0.001) and to 22.5 (3.1–89.6) mg/l in Child-Pugh C (P<0.01). Plasma samples of the cirrhotic patients showed significantly decreased levels of antithrombin, plasminogen, alpha-2-antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), significantly increased levels of tissue-type plasminogen activator (tPA) and no significant change in plasminogen activator inhibitor-1 (PAI-1). Spearman correlation tests in the whole group (controls plus patients) showed significant correlations between GFC and antithrombin (r=−0.44), plasminogen (r=−0.38), alpha-2-antiplasmin (r=−0.45), TAFI (r=−0.40), tPA (r=0.77) and PAI-1 (r=−0.51) (all p-values <0.0001). In conclusion, the new GFC test, which includes all components present in blood, indicates that the fibrinolytic capacity is strongly increased in patients with severe cirrhosis. The anti-fibrinolytic effects of decreased plasminogen levels in cirrhosis are apparently overruled by the pro-fibrinolytic effects of decreased alpha-2-antiplasmin and TAFI levels and particularly of increased tPA activity levels.
Mutational Analysis of VKOR.
Authors: Jin Da-Yun , Tie Jian-Ke , Stafford Darrel W.
Published: October 2019

More than 21 million prescriptions for warfarin are written yearly in the US for Vitamin K epoxide reductase (VKOR), the target of warfarin The vitamin K epoxide reductase, VKOR, apparently uses cysteines, 132 and 135 as active sites. In addition to cysteines 132 and 135, cysteines 43 and 51 are conserved throughout evolution. Rost et al. have mutated each of the cysteines in VKOR and found that, in whole cell lysates, mutations in of C43 result in less than 20% of wild-type activity while mutation of C53 eliminates activity. We have repeated these experiments and mutated all of the cysteine residues in VKOR. Our results in microsomes are similar to the results of Rost et. al. (2005) Thromb. Haemost. 94, 780–786. However, when the mutated enzymes are purified we find that the activity of C43 has 30% residual activity while C51 has 60% residual activity. Mutation of both residues in the same molecule results in an enzyme that is similar to the C51 mutation. In addition, we find that a portion of purified VKOR has a disulfide bond between residues 43 and 51. This suggested that we might be able to remove the loop between C43 and C51 and retain activity. Indeed, the mutated enzyme with this loop removed also has substantial activity. Warfarin inhibition studies suggest that these mutations do not materially affect warfarin sensitivity. Our results stress the importance of utilizing purified enzyme for interpreting the results of mutations in VKOR.
High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin-1β through the NFκ B, JNK and p38 MAP Kinase Signaling Pathways in Monocytes.
Authors: Khan Mohammad M. , Bradford Harland N. , Isordia-Salas Irma , Liu Yuchuan , Wu Yi , Espinola Ricardo G. , Ghebrehiwet Berhane , Colman Robert W.
Published: October 2019

Plasma high molecular weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to cleaved HK (HKa). We have reported that HKa releases cytokines (TNFα , IL-1β , IL-6) and chemokines (IL-8 and MCP-1) from isolated human peripheral blood monocytes. At a concentration of 600nM, Glutathione -S- transferase (GST) fusion proteins of kininogen domain 3 (D3), E7P - an active fragment of domain 3 (aaG255-Q292), HK domain 5 (D5), and D5 recombinant peptide HG (aa K420-D474) each stimulated secretion of IL-1β from monocytes. Receptors on monocytes including Mac-1, LFA-1, uPAR and gC1qR are required for IL-1β secretion from monocytes. We now report the signaling pathways and evidence for synthesis of IL-1 β in monocytes stimulated by HKa. Inhibitors of signaling pathways initiated by NFkB, JNK and p38, but not ERK, decreased IL-1b release from monocytes. A specific inhibitor of NFkB, MG-132 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 69.7, 67.3, 88.5% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 83.4% by MG-132 (100 μM). A specific inhibitor of JNK, SP 600125 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 55.7, 76.3, 78.9% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 90.23% by SP 600125 (100 μM). A specific inhibitor of p38, SB202190 (1,10 and 100 μM) significantly reduced IL-1β release from monocytes by 27.8, 14.2, 91.0% respectively, when stimulated by E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 76.8 % by SB202190 (100 μM). In contrast, the ERK activation inhibitor U0126 (1, 10 and 100 μM) failed to inhibit GST-E7P-induced release of IL-1β from monocytes but the release of IL-1β with LPS (10 μg/ml), was blocked by 77.4% at 100 μM. After monocytes (4 X 106/ml) were treated with HKa (600 nM) or LPS (10 ng/ml), total RNA was extracted and RT-PCR reactions for expression of IL-1β and gC1qR mRNA using specific primers were carried out. PCR products was separated in 4% ethidium bromide-stained agarose gels and photographed. No IL-1b mRNA was detected prior to exposure to HKa or LPS but both were detected at 1 hour. In contrast, gC1qR mRNA was present without stimulation by HKa. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by stimulating the synthesis and release of IL-1β from human monocytes using intracellular signaling pathways initiated by uPAR, β 2 integrins and gC1qR receptors.
The Ligand of Glucocorticoid-Induced TNF Receptor Is Expressed on Human Macrophages and Stimulates Proinflammatory and Anti-Tumor Effector Functions.
Authors: Kloss Mercedes , Krusch Matthias , Baltz Katrin M. , Peterfi Andrea , Kumbier Ingrid , Kanz Lothar , Salih Helmut R.
Published: October 2019

Macrophages are involved in inflammatory and anti-tumor responses by antigen presentation, release of immunoregulatory cytokines and cellular cytotoxicity. Among the molecules influencing macrophage functions are many members of the TNF superfamily. The TNF receptor family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and nuclear factor kappa B and abrogates suppression of murine but not human regulatory T cells. Expression of its cognate ligand GITRL has, in humans, been detected in dendritic cells and various healthy nonlymphoid tissues. Up to now, nothing is known regarding the function of human GITRL, while in mice, reverse signaling through GITRL has been shown to influence the activation and survival of macrophages. Here we report for the first time that GITRL is expressed in human macrophages as determined by FACS and RT-PCR analysis. Reverse signaling through GITRL using a recombinant GITR-Ig fusion protein differentially affects expression of costimulatory molecules like CD80, CD86 and B7-H1, death ligands like TNF, CD178 and TRAIL as well as MHC class I and II on the macrophage surface. Furthermore, stimulation of GITRL enhances phagocytosis and respiratory burst of macrophages as determined by analysis of ingestion of PE-labeled polystyrene microspheres and oxidation of dichlor-fluorescein diacetate, respectively by FACS. Interaction of GITRL with its receptor also leads to significantly increased production of proinflammatory cytokines like TNF, IL-6 and IL-8 as determined by ELISA. In addition, stimulation of GITRL significantly increases the killing of various human tumor cell lines by macrophages as determined by cytotoxicity assays. Taken together, our data demonstrate that GITRL plays an important role in the stimulation of macrophage-mediated inflammatory responses and immune responses against tumors.
Delineation of the Common Developmental Pathway for Granulocyte/Monocyte and Lymphoid Lineages by Using an Expression Reporter for PU.1.
Authors: Arinobu Yojiro , Mizuno Shin-ichi , Shigematsu Hirokazu , Ozawa Hidetoshi , Chong Yong , Iwasaki Hiromi , Kastner Philippe , Chan Susan , Akashi Koichi
Published: October 2019

Understanding how multipotent cells commit to each of their terminal fate potentials is an important aspect of stem cell biology. Hematopoietic stem cells (HSCs) of Lin −Sca-1+c-Kit+ (LSK) phenotype have been purified, which were further divided into CD34-long-term and CD34+ short-term (ST)-HSCs. The existence of phenotypically isolatable common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) downstream of ST-HSCs suggests that the first commitment step after the HSC stage is the bifurcation of lymphoid vs. myeloid pathway. Recent studies, however, suggest that the loss of MegE potential could be an early event in HSC stage. For example, LSK cells activating RAG-1 or Flt-3 expression retained granulocyte/monocyte (GM) but not megakaryocyte/erythrocyte (MegE) potential together with lymphoid potential, suggesting the existence of common progenitor for GM and lymphoid lineages. Here we report that a fraction of ST-LSK cells expressing high levels of PU.1, a transcription factor necessary for GM and lymphoid development, represents GM/lymphoid bipotent progenitors. In mice harboring knock-in GFP reporter for PU.1, LSK cells were divided into GFP high and GFP low subpopulations. Although PU.1low LSK cells were multipotent, PU.1high LSK cells differentiated only into GM and lymphoid cells in vitro and in vivo. We also found that single PU.1high LSK cells differentiated into GM, T and B cells in vivo. These data formally prove the existence of the third major early progenitor population activating PU.1 at a high level, the granulocyte/monocyte/lymphoid progenitor (GMLP). The existence of prospectively isolatable GMLPs strongly suggests that HSCs sequentially lose MegE then GM potential during their lymphoid commitment.
FGF-2 Supplementation Enhances Hematopoietic Differentiation of Rhesus ESC’s.
Authors: Shaaban Aimen , Gaur Lasya , Thomson James A. , Rajesh Deepika
Published: October 2019

Progress toward clinical application of embryonic stem cells (ESC) derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model such as the rhesus macaque. However, in contrast to human ESC’s (hESC’s), efforts to induce conclusive hematopoietic differentiation from rhesus ESC’s (rESC’s) have been unsuccessful. Despite their close phylogenetic relationship, subtle differences exist between the hematopoietic differentiation of rESC’s and hESC’s. We recently reported that although rESC’s have the potential for hematopoietic differentiation; they exhibit an arrest at the hematoendothelial precursor stage of hematopoietic development in culture conditions developed for hESC’s. One possible difference may be in the requirement for fibroblast growth factor (FGF) signaling. Despite documentation of its contribution to the maintenance ESC’s in an undifferentiated state, the role for FGF-2 in the hematopoietic differentiation of hESC’s and rESC’s has not been similarly examined. Given its critical role for the formation and subsequent hematopoietic differentiation of murine ESC-derived hemangioblasts, we wondered if enhanced hematopoietic differentiation from rESC’s could be achieved by culture supplementation with FGF-2. To answer this question, undifferentiated rESC’s were subjected to embryoid body (EB) differentiation with daily FGF-2 supplementation of the cytokine-rich media. Cultures were analyzed by flow cytometry after 16 days of EB culture. We found that the FGF-2 supplemented cultures appeared more robust with an overall higher numbers of cells. More importantly, a dramatic expansion of hematoendothelial precursors (Flk1hi+ VE-cadherin- CD45−), committed hematopoietic progenitors (CD34+CD45+Lin−), and hematopoietic cells (CD45+) was seen in FGF-2 supplemented cultures when compared to controls. These effects were consistent in two separate lines of rESC’s (R420 and R456). Next we wondered if the observed effect of FGF-2 on hematopoietic development was concentration-dependent. Therefore, we compared serial increases in FGF-2 concentration (0, 10, 50 and 100 ng/ml) of the EB differentiation media and found the effect to be concentration-dependent. From these results, we conclude that FGF-2 appears to play a critical role in the hematopoietic differentiation of rESC’s. Both the development of hematoendothelial precursors and the differentiation of committed hematopoietic cell types are augmented. To study this further, the significance of FGF signaling at various stages of rESC-derived hematopoietic differentiation must be evaluated. A better understanding of the requirements for FGF-2 in EB development will likely lead to improved protocols for the production of human and rhesus ESC-derived hematopoietic progenitors.