bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

AbstractThe 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.