bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nitrite Is Reduced to Nitric Oxide by Xanthine Oxidoreductase and Nitric Oxide Synthase in the Erythrocyte Membrane in Hypoxemia.
Authors: Rathod Krishnaraj S. , Webb Andrew J. , Lovell Matthew J. , Lecomte Florence , Ahluwalia Amrita 
Published: October 2019

Abstract
Evidence is emerging that inorganic nitrite, previously thought to be an inactive metabolite of nitric oxide (NO.), provides an alternative source of functionally active NO. under ischemic conditions when normal NO. synthesis, via nitric oxide synthase (NOS), is suppressed. Our previous studies have demonstrated that the reduction of nitrite to NO. in the ischemic heart is cardioprotective and dependent on xanthine oxidoreductase (XOR) activity (Webb et al., PNAS, 2004; 101:13683–13688). Recent evidence in erythrocytes suggests that nitrite is converted to NO. by a different mechanism involving deoxyhemoglobin (Cosby et al., Nat Med, 2003; 9:1498–1505). We hypothesized that erythrocytes may also contain XOR with nitrite reductase activity, and we investigated this possibility using suspensions of washed erythrocytes prepared from blood taken from healthy volunteers. Whole erythrocytes were incubated with nitrite (100 μM) in anaerobic conditions at pH 7.4 and at pH 6.8, mimicking normal physiology and severe acidosis respectively, and NO. production measured in parts per billion (ppb) using ozone chemiluminescence. Addition of erythrocytes to nitrite caused an elevation of NO. generation at pH 7.4 (0.28±0.06; p<0.05; n=6) and pH 6.8 (0.50±0.09; p<0.05; n=6) significantly above that produced by simple acidification (0.07±0.02 at pH 7.4, n=6; 0.17±0.06 at pH 6.8). NO. generation at pH 6.8 was enhanced by ~43% (P<0.01, n=6) in the presence of XOR substrate xanthine (10 μM) and attenuated by ~65% (P<0.05, n=6) by the XOR inhibitor allopurinol (100 μM). In contrast, xanthine and allopurinol did not alter nitrite-derived NO. generation at pH 7.4 (n=6). Treatment of erythrocytes with the NOS inhibitor L-NAME (300 μM) abolished nitrite-derived NO. generation at pH 7.4 but inhibited the response by only ~57% at pH 6.8 (n=6, P<0.05). Western blot analysis of erythrocyte membranes confirmed the presence of endothelial NOS (eNOS), as has been demonstrated recently (Kleinbongard et al., Blood, 2006; 107(7):2943–2951). Furthermore the presence of XOR in whole purified erythrocytes was confirmed both immunohistochemically and using immunoflorescence. In summary we have demonstrated that erythrocytes reduce nitrite to NO. through the enzymatic actions of XOR and eNOS. These mechanisms of NO. production complement that of deoxyhemoglobin and are likely to contribute to the protective effects of nitrite attributed to NO. generation in hypoxemic and ischaemic conditions.
An Erythoid-Specific Component of the Rhesus Stage Selector Protein, rNF-E4, Modulates γ-Globin Gene Expression Specifically.
Authors: Wu Ruiqiong , Desgardin Aurelie , Jane Stephen M. , Cunningham John M. 
Published: October 2019

Abstract
Understanding the molecular mechanisms that regulate γ-globin gene expression is essential for development of new therapeutic strategies for individuals with sickle cell disease and β-thalassemia. We have previously identified a tissue- and developmentally- specific multiprotein transacting factor complex, the human stage selector protein (SSP), which facilitates the interaction of the g-globin gene promoters with the upstream locus control region enhancer in fetal erythoid cells. This complex interacts with the stage selector element (SSE) in the proximal g-globin promoter, a regulatory motif phylogenetically conserved in primate species with a distinct fetal stage of β-globin like gene expression. Given these observations, we hypothesized that a similar complex modulates γ-globin in the rhesus macaque, a non-human primate model that has been utilized to study β-globin like gene expression. We focused our efforts on NF-E4, given that a human isoform of this factor confers erythroid and fetal specificity to the SSP complex. Fetal liver erythroblasts were obtained from rhesus embryos and analyzed by reverse transcriptase(RT)-PCR analysis for NF-E4 expression. NF-E4 like transcripts were identified in day 60, 80 and 120 embryonic erythroblasts, but not other rhesus tissues, demonstrating an erythroid-specific pattern of expression. Utilizing 5′ RACE, we cloned a full length NF-E4 transcript, identifying an open reading frame encoding a 131 amino acid polypeptide. This 20kD polypeptide shares a high degree of homology with human NF-E4, especially in its carboxy-terminal domain. Like human NF-E4, GST pulldown chromatography confirmed the ability of the rhesus factor to interact directly with CP2 and ALY, the other core components of the SSP. To evaluate rNF-E4 function in vivo, we utilized retrovirally mediated gene transfer to enforce expression of this factor in K562 cells, a model of human fetal erythropoiesis. Initial co-immunoprecipitation studies confirmed the in vivo interaction of rNF-E4 with other components of the SSP. Interestingly, we observed a specific 3-fold induction of γ-globin gene expression in rNF-E4 expressing cells when compared to controls. Moreover, we demonstrated that, like enforced expression of human NF-E4, rNF-E4 induced a significant increase in ε-globin gene expression. Taken together, our results suggest a conservation of NF-E4 expression and function in species with a fetal stage of globin gene expression. Moreover, the identification of rNF-E4 provides a platform for the pre-clinical development of therapeutic agents that induce high levels of NF-E4 in adult erythroblasts.
Conditional Disruption of the Murine Prothrombin Gene.
Authors: Mullins Eric S. , Flick Matthew J. , Kombrinck Keith W. , Degen Sandra J. , Sun William , Degen Jay L. 
Published: October 2019

Abstract
Thrombin is the core protease in the hemostatic system. Thrombin directs thrombus formation through the proteolytic conversion of fibrinogen to fibrin and the local activation of protease-activated receptors on platelets and other cells. In addition, thrombin controls the coagulation system through the activation of fXI, protein C and other key hemostatic factors. Interestingly, the biological role of thrombin is seemingly not limited to the maintenance of vascular integrity. There is appreciable evidence that thrombin-mediated proteolysis plays an important role in development, the inflammatory response, tissue repair, tumor cell metastasis and other physiological and pathological processes. Unfortunately, the embryonic and perinatal lethal phenotype previously described in prothrombin null (fII−/−) mice limited the utility of those knockout animals in better defining the larger role of fII in vivo. In order to develop the means to explore the importance of thrombin in disease processes within adult animals, a mouse line was generated carrying a conditional (“floxed”) fII knockout allele (fIIfx mice). Homozygous fIIfx/fx mice and compound heterozygous mice carrying one fII floxed allele and one fII null allele (fIIfx/− mice) developed to term, were present in offspring in the expected Mendelian frequencies, survived to adulthood and retained normal reproductive success. In the absence of Cre-mediated recombination, fIIfx/− mice maintained circulating fII levels that were low (approximately 10% of normal), but spontaneous bleeding events were never encountered in these animals. Studies of fIIfx/− mice carrying a Cre recombinase transgene known to be constitutively expressed in the liver showed that prothrombin levels can be reduced to levels incompatible with post-natal survival. More sophisticated studies using the polyI:C-inducible Mx-Cre system revealed that unchallenged Mx-Cre+/fIIfx/− mice consistently survived to adulthood. However, induction of Cre under conditions that result in near-complete recombination of target floxed alleles within the liver resulted in the development of spontaneous bleeding events and death within 7 days. Multiple sites of hemorrhage were evident in these challenged adults, including lower gastrointestinal and intracranial sites of bleeding. Immunological analysis of plasma collected from these animals revealed that they carried levels of fII below current detection limits (< 1% of normal). Studies are underway to establish both the lowest level of plasma fII compatible with long-term survival and define the effects of extremely low fII levels on disease processes in vivo.
Missense Mutations in the Proline-Rich Region of Coagulation Factor XII in Hereditary and Idiopathic Angioedema.
Authors: Dewald Georg , Bork Konrad 
Published: October 2019

Abstract
Hereditary angioedema (HAE) is characterized by recurrent skin swelling, abdominal pain attacks, and potentially life-threatening upper airway obstruction. The two classic HAE types (I and II) are caused by mutations in the complement C1 inhibitor gene, resulting in a functional deficiency of C1 inhibitor. Recently, a novel type of hereditary angioedema has been described, these patients showing normal C1 inhibitor concentration and activity in plasma (HAE with normal C1 inhibitor, HAE type III). With few exceptions, all reported patients have been women, and exposition to estrogens appears to be an important precipitating factor. Screening of twenty unrelated female patients with this new type of HAE for mutations in the coagulation factor XII gene revealed two different missense mutations, both located in exactly the same position within exon 9, namely in the second position of the codon (ACG) encoding Thr309 of the mature protein. Five patients showed a C→A transversion (1032C→A), predicting a threonine-to-lysine substitution (Thr309Lys); one additional patient showed a C→G transversion (1032C→G), resulting in a threonine-to-arginine substitution (Thr309Arg). Thus, in 6 of 20 unrelated patients the wild-type threonine residue is substituted by a basic amino acid residue. The mutations are located in the proline-rich region of factor XII which may play some role in the binding of factor XII to negatively charged surfaces, and, thus, may eventually influence mechanisms of contact activation. The predicted structural and functional impact of the mutations, their absence in healthy control individuals (n=145), and their co-segregation with the phenotype in five families with altogether 20 affected women provide strong support that they cause disease (Biochem. Biophys. Res. Commun.343: 1286–1289, 2006). We speculated if these mutations may also be present in singular angioedema cases, i.e., patients with no affected family members. We therefore studied 58 such patients with idiopathic recurrent angioedema (23 men, 35 women) by sequencing of exon 9 of the F12 gene. In two patients, both female, the 1032C→A (Thr309Lys) mutation was observed. Thus, this mutation may also play a role in a subgroup of patients with idiopathic angioedema, in particular in female patients. The known estrogen-dependent regulation of factor XII expression may well be seen in association with the disease manifestation mainly in women.
hFlt3 Expression Marks Long-Term Hematopoietic Stem Cells, but Not the Pathway Common to Granulocyte/Monocyte and Lymphoid Lineages in Human Hematopoiesis.
Authors: Kikushige Yoshikane , Yoshimoto Goichi , Miyamoto Toshihiro , Ishikawa Fumihiko , Iwasaki Hiromi , Harada Mine , Akashi Koichi 
Published: October 2019

Abstract
Flt3 is a tyrosine kinase receptor, whose signals support proliferation and differentiation of early hematopoietic progenitors in cooperation with other cytokine signals. The concept of myeloid vs. lymphoid commitment in murine hematopoiesis has been challenged based on the analysis of Flt3 expression (Adolfsson et al, Cell, 2006): Long-term murine hematopoietic stem cells (HSCs) with self-renewal activity do not express murine Flt3 (mFlt3), but mFlt3 is upregulated in a fraction of progenitors common to granulocyte/monocyte and lymphoid lineages, suggesting the existence of the 3rd pathway of murine hematolymphoid development. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 99, 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. Surprisingly, all single human hCD34+hCD38-Thy-1+Lin-long-term HSCs expressed Flt3 at a low level. Purified Flt3lo HSCs can reconstitute NOD-scid/IL2Rγnull newborns (Blood 106, 2005) for a long-term after adoptive transfer of only 100 cells, and generated all hematopoietic cells including GM, megakaryocyte/erythrocyte (MegE), T, B and NK cells, providing formal proof that hFlt3 is expressed in long-term multipotent human HSCs. In the myeloid pathway, hCD34+hCD38+hCD45RA-IL-3RαloLin-CMPs expressed low to negative levels of hFlt3, and hCD34+hCD38+CD45RA-IL-3Rα-Lin-MEPs did not express Flt3. However, in marked contrast to murine progenitors, hCD34+hCD38+hCD45RA+IL-3RαloLin-GMPs uniformly expressed hFlt3 at the highest level. We then separated CMPs into 2 fractions with negative and low level expression of Flt3. In vitro culture in the presence of cytokine cocktail revealed that hFlt3lo CMPs differentiated into hFlt3-CMPs, hFlt3-MEPs and hFlt3hi GMPs, while hFlt3-CMPs mainly generated hFlt3-MEPs in vitro, suggesting that hFlt3lo CMPs is located upstream of the hFlt3-CMP stage that has skewed differentiation potential into the MegE lineage. In vitro differentiation activity of these progenitors was not affected with or without hFlt3 ligand in the culture. hFlt3 ligand, however, appeared to be able to maintain survival of hFlt3-expressing stem and progenitor cells at least in vitro. Thus, in striking contrast to mice, hFlt3 expression is already initiated at the most primitive long-term HSC stage, and is progressively upregulated in accordance with GM lineage differentiation. These data collectively suggest that the role of Flt3 signaling in hematopoietic development might be quite different between human and mouse, and that hFlt3 signaling may especially play a critical role in maintenance of long-term HSCs and in development of GM cells in human hematopoiesis.
SLAM-Family Expression on Human Mobilized Peripheral Blood Progenitors.
Authors: Savona Michael R. , Kiel Mark J. , Leavitt Andrew D. , Morrison Sean J. 
Published: October 2019

Abstract
Background and significance: A simple but precise method to identify hematopoietic stem cells within mobilized peripheral blood would be useful for transplantation. Our lab has recently identified a family of surface markers whose differential expression distinguishes mouse hematopoietic stem cells from other hematopoietic progenitors. The founding member of the signaling lymphocyte attractant molecule (SLAM) family, CD150, was expressed on all hematopoietic stem cells (HSCs) but not on other hematopoietic progenitors. Other SLAM-family members, including CD244 and CD48, were expressed by non-self-renewing multipotent progenitors and most colony-forming restricted progenitors, respectively. As a result, mouse stem cells can be highly purified as CD150+CD48− cells, dramatically simplifying and improving the purification of mouse HSCs. To begin to test whether SLAM family markers can facilitate the identification and purification of human hematopoietic stem cells, we have assessed the frequency of CD150+CD48− cells in mobilized peripheral blood and compared their distribution to that of CD34+CD38− cells, which are known to be highly enriched for human hematopoietic stem cells.
Methods: Mobilized human peripheral blood samples were stained with anti-CD150 (conjugated to the FITC), anti-CD48 (PE), anti-CD41 (PE), anti-CD34 (APC), and anti-CD38 (PE-Cy5) antibodies. Samples were analyzed by flow-cytometry.
Results: We have identified a population of CD150+CD48−CD41− cells within human mobilized peripheral blood that is present at a similar frequency as the same population in mobilized mouse peripheral blood (mean 0.039±0.11%). The CD34+CD38− population was similarly infrequent. Interestingly, 16.3±19.5% of CD150+CD48−CD41− cells were also CD34+ whereas only 1.13±3.45% of the CD34+CD38− population was CD150+CD48−CD41− raising the possibility that SLAM-family members may substantially improve the purity of human hematopoietic stem cells.
Conclusion: Murine and human hematopoietic tissues have a similar frequency of CD150+CD48−CD41− cells. It is possible that the use of SLAM-family markers might enhance the identification and purification of human hematopoietic stem cells beyond what is possible using CD34 and CD38. We are currently performing reconstitution assays to test this functionally.
Peripheral Blood Mononuclear Cells Peripheral Blood Mononuclear Cells
Characterization of Placenta Derived Adherent Cells (PDAC): A Novel Type of Stem Cells Isolated from Human Placenta.
Authors: Edinger James , Ye Qian , Paludan Casper , Wang Jia-Lun , Hofgartner Wolfgang , Hariri Robert 
Published: October 2019

Abstract
The placenta is a rich source of ethically non-controversial stem cells. In this work, we describe the development of procedures to isolate and culture adherent stem cells from a full term, postpartum human placenta (Placenta Derived Adherent Cells, PDACs). PDACs are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, cord, placental cotyledons, or any combination thereof. PDACs were established in a medium containing low concentrations of fetal calf serum and limited growth factors. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70%. We found that PDACs differentiate down the adipocyte, chondrocyte and osteocyte lineages. In an induction medium containing IBMX, insulin, dexamethasone and indomethacin, PDACs turned into fat laden adipocytes in 3 to 5 weeks. Under osteogenic induction culture conditions, PDACs were found to form bone nodules and have calcium depositions in their extracellular matrix. Chondrogenic differentiation of PDACs was performed in micropellets and was confirmed by formation of glycosaminoglycan in the tissue aggregates. In addition, we showed that PDACs suppress an MLR consisting of suitable effectors (T cells or NK cells) and target populations (allogeneic irradiated PBMCs or mature DCs). The range of suppression of T cell proliferation was 50 to 95 %. The observed suppression of the MLR is likely cell-to-cell contact dependent in the range of 20%. In summary, the PDACs described herein exhibit in vitro properties of pluripotent stem cells. The placenta is an excellent raw material for the isolation of stem cells.
Marrow Makes Thyroid but Not Adrenal Tissues in Humans.
Authors: Davenport Jake , Jorgensen Marda L. , Scott Edward W. , Cogle Christopher R. 
Published: October 2019

Abstract
Human marrow has shown potential to contribute to organ various organs. We previously demonstrated marrow contribution to the brain in humans, without evidence for fusion. Given the observations of marrow contributing to the other end-organs, we questioned whether marrow also repair endocrine glands like the thyroid and adrenal glands. To address this question, we identified thyroid (n=3) and adrenal tissues (n=3) obtained from women who received hematopoietic cell transplantation from male donors. Thyroid tissues were stained for thyroid peroxidase (TPO), CD45, and XY FISH. Adrenal tissues were stained for synaptophysin, CD45, and XY FISH. Analysis of 450 thyroid follicles (150 per patient) found 2% to 5% of follicles containing a male/marrow-derived thyroid follicle demonstrating follicular orientation, TPO expression, lacking CD45, and containing a Y chromosome. The median number of male/marrow-derived cells in follicles was 1. None of the male/marrow-derived cells demonstrated a fusion karyotype (XXY or XXXY). In contrast, adrenal tissues analyzed in the women demonstrated no male cells throughout all layers. In conclusion, our results show that human marrow can contribute to thyroid follicles and express thyroid proteins, however at a modest level. Furthermore, adrenal tissues in these women demonstrated no evidence of marrow contribution. These results expose a potential strategy of thyroid repair by illuminating the potential of marrow contribution to thyroid epithelia.
Substrate Preferences for Antiplasmin Cleaving Enzyme Hydrolysis of Peptides Derived from the α2-Antiplasmin Sequence.
Authors: Jackson Kenneth W. , Christiansen Victoria J. , Lee Kyung N. , Mason Christina F. , McKee Patrick A. 
Published: October 2019

Abstract
Antiplasmin cleaving enzyme (APCE) is a proteinase that specifically, but slowly cleaves the Pro-Asn bond in long-form α2-antiplasmin (Met-α2AP) in human plasma. This slow cleavage produces a steady-state plasma mixture of Met-α2AP and an N-terminally shortened form of antiplasmin, Asn-α2AP. The Asn-α2AP form crosslinks to fibrin ~13-fold faster than Met-α2AP. A faster crosslink rate causes a greater number of antiplasmin molecules to become bound during fibrin formation, thereby enhancing resistance to fibrinolysis. Inhibition of plasma APCE may decrease the number of antiplasmin molecules crosslinked and result in clots that are more easily removed during fibrinolysis. Therefore, an inhibitor specific for APCE could potentially be used to regulate fibrinolysis. Human Met-α2AP exists in two polymorphic forms at position six in the mature sequence, with arginine predominant, and tryptophan accounting for a lesser percentage. We have determined the relative cleavage rates of synthetic peptides from a peptide library that span the cleavage site. The peptides contained all common amino acids except cysteine in the polymorphic position (P7 position). Arg was the optimal amino acid in this position with a relative cleavage rate ~5–10-fold faster than other amino acids except Lys, which was ~70% of the Arg rate. The P7 position Arg enhancement was also observed when Arg was in the P6 or P5 position, but no enhancement was observed when Arg was moved to positions P8, P4, P3 or P2. It was also determined that APCE is preferentially an endoproteinase rather than an aminodipeptidase, with a 10-fold greater rate of hydrolysis of the internal Pro-Asn bond in the Met-α2AP 1–17 peptide sequence MEPLGRQLTSGP-NQEQV over the Pro-Asn bond penultimate to the amino-terminal bond in the Met-α2AP 11–27 peptide sequence GP-NQEQVSPLTLLKLGN in peptide hydrolysis experiments. We conclude that APCE inhibitors designed with a positive charge placed upstream of the Pro-X scissile bond equivalent to five to seven amino acid residues may prove to be highly potent and specific. In addition, such inhibitors should also prove useful for blocking the activity of the closely related enzyme fibroblast activation protein. This work was supported by NIH grant HL072995.