bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clathrin-Mediated Endocytosis of Soybean Ferritin by Caco-2 Cells.
Authors: Walter Tomas , Martin Carol San , Theil Elizabeth C. , Pizarro Fernando , Garri Carolina , Nunez Marco T. 
Published: October 2019

Abstract
Background: The mechanisms of intestinal absorption of non-heme and heme iron have been extensively studied. However ferritin, which has been shown to be a good source of bioavailable iron in the diet, could have a different mechanism.
Methods: Here we examined cellular mechanisms by which recombinant soybean ferritin (rSBFn) is absorbed in a cell model for intestinal epithelia. The internalization of mineralized (480 Fe) rSBFn, labeled with radioactive iodine, or the fluorescent probe Oregon Green®, was monitored in cultured, polarized Caco-2 cells. Additionally, internalization of rSBFn iron into the labile iron pool was determined by calcein fluorescence quenching.
Results: Caco-2 cells internalized 131 I-rSBFn from the apical border in a temperature, concentration and time dependent fashion. The apical membrane showed saturable binding sites with a KD = 3.43 ×10−9 M. After internalization, rSBFn was degraded, increasing iron in the cytosolic, labile pool, analyzed as calcein quenching. Horse spleen ferritin also induced calcein quenching. The amount of iron incorporated from 5 nM rSBFn was similar to that contributed by micromolar amounts of heme and non-heme iron. rSBFn endocytosis was inhibited by treatment with short RNAi for the μ 2 sub-unit of the assembly peptide 2 complex, by cytosol acidification and by hyperosmolarity, which all inhibit clathrin-mediated endocytosis. Filipin II, an inhibitor of caveolar endocytosis, had no effect.
Conclusion: These results suggests the presence of a third route of intestinal iron absorption in which ferritin from plant or animal sources is internalized from the apical medium of enterocytes by a clathrin-dependent endocytic process.
Acyl-CoA Turnover in RBC.
Authors: Soupene Eric , Siliakus Melvin , Kuypers Frans A. 
Published: October 2019

Abstract
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). While formation and utilization of acyl-CoA takes place at the membrane, Acyl-CoA Binding Domain containing proteins (ACBD) in RBC cytosol bind acyl-CoA, limiting product feedback inhibition on ACSL and distribute Acyl-CoA to the various acyl-utilizing enzymes while protecting the cells against its potent detergent character.
We have identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC, a protein with several isoforms that acts as a dimer. To relate its structure to activity, we report the expression of different modified forms in E. coli. Our data indicate that, despite the observed activity, enzyme studies of these mammalian membrane proteins in the host E. coli are strongly hampered by their aggregation into inclusion bodies. While activity can be measured, data on enzyme kinetics and specificity are questionable. Oleoyl-CoA formation from oleic acid, CoA and ATP reveled that the two transmembrane spanning segments predicted at the amino-terminus of the protein are not essential for its activity. Moreover, they are not essential for dimer formation and strong association with membranes. ACSL6 appears to be an integral membrane protein. One of the five spliced isoforms of ACSL6 reported, lacks the so-called fatty acid Gate-domain, and appears to be unable to activate long chain fatty acids. We hypothesize that this form modulates activity of the other active isoforms of ACSL6 through hetero-dimer formation. An EST clone of erythroid precursor cells identified ACBD6 as a potential AcylCoA binding protein in RBC. This modular protein contains an Acyl-CoA binding domain at the amino-terminus and two Ankyrin-repeat motifs (ANK) at the C-terminus. Both the full-length protein and the N-terminus domain were soluble when expressed and purified in E. coli. Expression of the C-terminus domain by itself rendered an insoluble protein. We report that ACBD6 binds long-chain acyl-CoA with a preference for C18:1-CoA over C20:4 and C16:0-CoA and does not bind fatty acid. Truncation of the ANK domain had no effect on the binding activity of the N-terminus domain. Together our findings implicate ACBD6 as part of the Acyl-CoA turnover mechanism in RBC, its actual role on the kinetics of ACSL and/or LPLAT activity needs to be established. The description of these proteins involved in Acyl-CoA turnover in RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.
A Facilitated Tracking and Transcription Mechanism of the HS2 Enhancer in the β-Globin Locus Control Region.
Authors: Tuan Dorothy , Zhu Xingguo , Ling Jianhua , Zhang Ling , Pi Wenhu , Wu Min 
Published: October 2019

Abstract
The 100 kb human β-globin gene locus contains the embryonic ε-, the fetal Gγ- and Aγ-, and the adult δ- and β-globin genes arranged in the transcriptional order of 5′ ε-Gγ-Aγ-δ-β 3′. The β-globin Locus Control Region (LCR), defined by DNase I hypersensitive sites HS1, 2, 3, 4 and 5 located far upstream of the globin genes, is absolutely required for transcriptional activation of the globin genes in erythroid cells. The HS2 site, located respectively 11 and 55 kb upstream of the ε- and β-globin genes, possesses prominent enhancer activity and is able to activate transcription of the globin genes over the long distance. How the HS2 enhancer acts over the long distance is not fully understood. Three mechanisms have been proposed to explain long-range enhancer function: The looping model, the protein tracking/linking model and the facilitated tracking model. We have shown earlier that a transcription mechanism of the RNA polymerase II (pol II)-complex assembled by the HS2 enhancer mediates long-range HS2 enhancer function. In the present study, we used the chicken HS4 insulator to further study the mechanism of long-range HS2 enhancer function. We created the LCR(+I) and LCR(−I) lines in human erythroid K562 cells using Cre-loxP mediated in situ recombination and in zebrafish. The integrated LCR(+I) and LCR(−I) plasmids spanned the natural 11 kb human ε-globin gene locus either with or without the chicken HS4 insulator inserted between the LCR HS2 enhancer and the ε-globin promoter. Analysis of enhancer function in the integrated plasmids and the endogenous ε-globin gene locus provided evidence for a novel facilitated tracking and transcription mechanism of long-range enhancer function: The HS2 enhancer complex containing both the enhancer DNA and the associated pol II tracked and transcribed through the 10 kb intervening DNA to loop with and activate the ε-globin promoter. The interposed insulator interrupted this facilitated tracking and transcription mechanism of the enhancer complex through the intervening DNA, thereby blocking long-range enhancer function.
The Natural History of the Neutrophil Elastase and Coagulation Factor Response to E.coli in a Baboon Model of E.coli/Sepsis Suggests That Elevated Neutrophil Elastase Activity Is Sustained Regardless of Outcome in Clinical Sepsis.
Authors: Samis John A. , Stewart Kenneth A. , Taylor Fletcher B. , Fournier Daniel J. , Nesheim Michael E. 
Published: October 2019

Abstract
Neutrophil elastase (NE) functions in the destruction of gram negative bacteria. NE consumption/inactivation of coagulation factors may contribute to the coagulopathies that accompany infections upon insuffcient regulation by its primary plasma inhibitor, alpha-1-protease inhibitor (A1PI). The temporal relationships between NE levels, A1PI activity and antigen, and the activity of Factor (F) II, V, VII, VIII, IX, X, and fibrinogen were studied in three baboons after infusion of 2.40 x 106 cfu/kg (low/sublethal), 1.05 x 108 cfu/kg (medium/sublethal), and 6.49 x 108 cfu/kg (lethal) of E.coli. Immunoblotting for A1PI under native conditions indicated that the A1PI/NE complex levels increased by 5- to 93-fold after 24hrs and remained 3- to 580-fold above baseline at 72hrs and 53hrs following sublethal and lethal E.coli challenge, respectively. The A1PI specific activity (activity/antigen) decreased by 55% and 30% after 48–72hrs and 48–53hrs following the medium and lethal dose, respectively. Increased amounts of 2 forms of A1PI resolved by native PAGE/immunoblotting correlated with decreased inhibitor specific activity. At the lethal dose of E.coli, decreases (%) in fibrinogen (96%), FV (60%), and FVIII (60%) occurred within 2–6hrs. By 53hrs, FV and FVIII levels returned to normal and fibrinogen was twice normal. Similar changes of smaller magnitudes occurred at the lower doses of E.coli. At the lethal dose of E.coli, decreases in FII (50%), FVII (80%), FIX (80%), and FX (60%) occurred over the first 24hrs and these levels were sustained through 53hrs. Similar changes of smaller magnitudes occurred at the lower doses. In conclusion, there is a temporal association between coagulation factor consumption/inactivation, appearance of the A1PI/NE complex, and A1PI inactivation during E.coli/sepsis in baboons. The marked elevation of the A1PI/NE complex beyond 24hrs, even after sublethal challenge, indicates for the first time that increased neutrophil and NE activities persist beyond the initial inflammatory and coagulopathic stages into the subsequent cell injury and degeneration stages that terminate in either recovery or death from E.coli/sepsis.
Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.
Authors: Patel Sanjay , Berry Leslie R. , Smith Lesley , Chan Anthony 
Published: October 2019

Abstract
The rate of prothrombin to thrombin conversion by factor Xa (Xa) is enhanced when Xa is incorporated into the surface-bound prothrombinase complex. However, in comparison to the free state, Xa within the prothrombinase complex is afforded protection from antithrombin + heparin (AT+H) inactivation. We have shown that, unlike AT+H, a covalent conjugate of AT and H (ATH) can neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa +/− prothrombinase complex. Mixtures of CaCl2, phospholipid vesicles, factor Va (Va) and prothrombin in TSP buffer, were combined with Xa. Following addition of either AT+H or ATH, time samples were neutralized with Na2EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k2) were calculated from plots of activity versus time. Results were compared to corresponding experiments with Xa alone. AT+H inactivation of Xa in prothrombinase occurred at a k2 (x 107 M−1min−1) of 2.34 +/− 0.09. In contrast, neutralization of free Xa by AT+H was significantly faster (k2 = 8.34 +/− 0.18, p = 0.03). Reaction with ATH showed no significant rate difference for Xa inhibition in either the complexed or free states (18.5 +/− 3.3 and 16.3 +/− 3.7, respectively). Intriguingly, the rates achieved for ATH inhibition of complexed and free Xa were significantly greater than that for AT+H with free Xa (p=0.03 and p=0.02, respectively). We conclude that covalent complexes of AT and H do not encounter resistance in the inhibition of Xa in prothrombinase, as seen for non-covalent AT+H mixtures. Thus, it is possible for ATH to effectively inhibit the propagation phase of thrombin generation and thus dampen thrombin production via neutralization of Xa in prothrombinase.
Inhibition of Heparinase I by Defibrotide with Potential Clinical Implications.
Authors: Fareed Jawed , Sonevytsky Adrian , Iqbal Omer , Jeske Walter P. , Iacobelli Massimo , Hoppensteadt Debra 
Published: October 2019

Abstract
Defibrotide represents a poly-deoxyribonucleotide derived antischemic and antithrombotic agent. Currently this agent is used for the management of transplantation associated with vascular complications. Defibrotide is a polyanionic electrolyte capable of releasing endogenous antithrombotic mediators such as (tissue factor pathway inhibitor (TFPI) and heparans. Co-administration of defibrotide with heparin has been shown to produce an augmentation of the effect of heparin and an increase in the biologic half-life. This may be due to the drug interactions or inhibition of endogenous heparin digesting enzyme by defibrotide. To test the hypothesis that defibrotide may inhibit heparin digesting enzymes such as heparinases (I–III), the effects of defibrotide were investigated on the digestion of unfractionated heparin. Unfractionated heparin was subjected to bacterial heparinase I (flavobacterion heparinicum) in an isolated biochemical systems where unfractionated heparin was used as a substrate at a fixed concentration of 750 μg/ml. Graded amounts of defibrotide in the range of 6.25–250 μg/ml were supplemented to this mixture. The degree of heparinase digestion of unfractionated heparin was determined by using high performance liquid chromatographic methods and computation of oligosaccharide profiles. Heparinase I was found to digest heparin (MW=15.2kDa) converting it to low molecular mass heparins (MW=4.1kDa). At concentrations of >125 μg/ml defibrotide produced an almost complete inhibition of heparinase I digestion. This inhibition of heparinase digestion was dependent on defibrotide concentration and the IC50 was found to be 12.5 μg/ml. These results suggest that polyelectrolytes such as defibrotide are capable of inhibiting heparin/heparan digestion enzymes. The observed potentiation of anticoagulant effect of heparin in patients treated simultaneously with defibrotide may partially be due to the inhibition of endogenous heparin digesting enzymes. Subsequent studies in primates have revealed that the anticoagulant effects of both unfractionated heparin and low-molecular mass heparins are potentiated by defibrotide as determined by AUC measurements for the circulating level of these agents. Thus, these interactions should be taken into account to optimize anticoagulant management where these drugs are used in a combination regimen.
New Role for ATP P2X1 Receptors in the Control of Neutrophil Apoptosis and Respiratory Burst Activity.
Authors: Faccinetto Celine , Lecut Christelle , Greimers Roland , Bours Vincent , Oury Cecile 
Published: October 2019

Abstract
P2X receptors are membrane non-selective cation channels that open in response to the binding of extracellular ATP. Seven genes encode P2X receptor subunits (P2X1–7) in vertebrates. Channels form homo- or hetero-multimers of several subunits that are highly permeable to Ca2+. P2X receptors are widely distributed. In recent years, increasing attention has been paid to extracellular ATP as a candidate danger signal locally released at the inception of inflammation. One of the most striking features of ATP is its ability to promote P2X7 receptor-mediated massive release of mature IL-1β from LPS-primed mononuclear phagocytes. On neutrophils, ATP rises intracellular Ca2+ concentrations, contributes to degranulation, adhesion, oxidative burst and delays apoptosis, events that may partly depend on the G-protein coupled P2Y2 receptor and on P2X7. In the present work, RT-PCR, Western blotting and immunofluorescence experiments reveal that neutrophils also express P2X1 and P2X5 receptor subtypes. A microarray analysis indicates that a 3-hour treatment of human peripheral blood neutrophils with the selective P2X1 and P2X1/5 receptor agonist, a,β-meATP, changes the expression of genes mainly involved in the control of cell fate. Accordingly, this agonist causes an increase of phosphatidylserine exposure on neutrophil membranes, maximally occurring after 3 hours and lasting until 18 hours of culture. In the presence of the protein synthesis inhibitor cycloheximide, a,β -meATP promotes caspase-3-dependent neutrophil apoptosis after 3 hours, which is correlated with highly reduced Fcγ RIII (CD16) membrane expression. In addition, a 1 min pretreatment of neutrophils with a,β -meATP potently increases tumor necrosis factor-a (TNF-α )-driven priming (30 min) of the respiratory burst induced by the bacterially derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Furthermore, a,β -meATP produces L-selectin (CD62L) shedding by its own and in an additive manner with TNF-α . This agonist also induces rapid and reversible phosphorylation of the survival kinases ERK (starting after 2 min) and Akt (15 min) as well as phosphorylation and degradation (after 2 min) of I-κ Bα , an inhibitor of the anti-apoptotic transcription factor NF-κ B. Hence, neutrophil P2X1 receptors might have a dual role in inflammation; they would both contribute to neutrophil activation and promote cell death of neutrophils that have reached the end of their useful life span. Activation of P2X1 receptors by extracellular ATP may thus represent novel regulatory mechanisms that govern neutrophil function.
Identification and Characterization of Zebrafish Mast Cells.
Authors: Berman Jason N. , Seibert Jake , Crosby Amanda , Fraser Robert , Gritsenko Olga , Pohajdak Bill , Lin Tong-jun 
Published: October 2019

Abstract
Mast cells (MCs) are well-known for their role in allergic reactions and inflammation, but their developmental origin is controversial. In addition, abnormal clonal proliferation of MCs, referred to as systemic mastocytosis (SM), may be associated with acute myeloid leukemia, portending a poor outcome. Mutations in the C-KIT tyrosine kinase have been identified in SM, that can be potentially targeted by small molecule tyrosine kinase inhibitors. The zebrafish is a robust model organism for studying hematopoiesis and leukemogenesis, and has an inherent capacity to accommodate genetic and chemical modifier screens. Thus, this system holds potential for elucidating MC lineage and for use in high-throughput screening of targeted therapeutics in MC diseases. MCs have not been previously described in zebrafish. We have identified putative MCs in adult zebrafish gill and intestine containing eosinophilic granules that stain with peroxidase acid shift (PAS) and toluidine blue, as well as with carboxypeptidase A5 (cpa5), a zebrafish homologue of the mammalian MC specific enzyme, CPA1. Electron microscopic analysis demonstrates a striking morphologic resemblance to mammalian MCs with abundant homogeneous dense granules. Classical functional studies reveal degranulation of these cells and increased histamine production upon stimulation with compound 48/80 and stimulated IgE. Whole mount in situ hybridization experiments on zebrafish embryos demonstrate cpa5 expression in a population of blood cells at 28 hours post-fertilization co-localizing with the early myeloid marker, pu.1, the granulocytic marker, mpo, and monocytic markers, l-plastin and lysozyme C. These data point to the existence of a zebrafish MC equivalent and suggests that this lineage arises at the level of the granulocyte/monocyte progenitor. Ongoing characterization of these cells will provide further insight into the mechanisms underlying MC development. Furthermore, a zebrafish cpa5 promoter element has been cloned and is being used to generate transgenic zebrafish lines. These transgenic zebrafish will provide a valuable tool in identifying new effective therapeutic agents targeting MCs in allergic and immune responses, as well as in their contribution to leukemic progression.
Novel Insights into the Maturation and Enucleation of Primitive Erythroblasts.
Authors: Baron Margaret H. , Fraser Stuart T. , Isern Joan 
Published: October 2019

Abstract
Primitive erythroblasts (EryP), the first blood cell type to form during mammalian embryogenesis, arise in the yolk sac (YS) as a synchronous cohort before the establishment of the embryonic circulation. The large, nucleated EryP begin to circulate around midgestation, when connections between YS and embryo mature. Two to three days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryP within the circulation. Cells of the two erythroid lineages differ not only in size but also in their expression of globin genes. Recently it has been shown that EryP begin to enucleate around E12.5 in the mouse. We have begun to characterize EryP maturation during mouse embryogenesis, with a focus on circulating cells. Analysis of embryonic blood at different stages revealed a stepwise developmental progression within the primitive erythroid lineage: loss of nucleoli (E9.5–10.5); decrease in cell diameter and cross-sectional area (E10.5–11.5); progressive nuclear condensation (E10.5 onwards); and enucleation (E12.5 onwards). Thereafter, EryD are also present in the bloodstream. A major obstacle to the study of primitive erythroid development is the current inability to easily distinguish EryP from EryD and to cleanly separate these populations, particularly given that enucleation can no longer be considered an exclusively definitive erythroid characteristic. No EryP-specific surface antigens have been identified that would permit the detection and isolation of cells of this lineage. We have exploited the restricted expression of the human embryonic beta-like globin gene epsilon to generate EryP lineage-specific transgenic mouse and embryonic stem (ES) cell models in which green fluorescent protein (GFP) reporter expression is driven exclusively in EryP and have used this line to track quantitatively the differentiation and maturation of circulating EryP during embryogenesis. The maturation of primitive erythroblasts was reflected in their dynamic expression of Ter119 and CD71. Up-regulation of a number of nonerythroid-specific cell surface markers, including integrins and other adhesion molecules, was apparent beginning around E12.5, reflecting a previously unrecognized EryP maturation process. Interestingly, enucleation of EryP is accompanied by changes in surface antigen expression. Thus, the phenotype of primitive reticulocytes is distinct from that of their nucleated precursors. In contrast with the prevailing view that EryP are present only transiently in the embryo, we find that they comprise a stable cell population that persists through the end of gestation. The human epsilon-globin::GFP transgenic mouse model provides a novel system for defining, at the cellular and molecular level, the mechanisms involved in development of the primitive erythroid lineage.