bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multi-Step Fibrinogen-αIIbβ3 Binding/Unbinding Revealed at the Single Molecule Level Using Laser Tweezers.
Authors: Litvinov Rustem I. , Shuman Henry , Bennett Joel S. , Weisel John W. 
Published: October 2019

Abstract
The regulated ability of the platelet integrin αIIbβ3 to bind fibrinogen plays a crucial role in platelet aggregation and primary hemostasis. We have developed a model system based on laser tweezers that enables us to measure directly the mechanical forces needed to separate single receptor-ligand complexes. In this system, a ligand-coated bead can be trapped by the laser and repeatedly brought into contact with a receptor-coated pedestal so that the forces required to separate the two can be measured and displayed as “rupture force” histograms. We have applied this system to the interaction of fibrinogen and αIIbβ3 by measuring the rupture force required to separate a laser-trapped bead coated with fibrinogen from an immobilized pedestal coated with purified αIIbβ3. These measurements revealed that bimolecular αIIbβ3-fibrinogen interactions are characterized by rupture forces ranging from 20 pN to 150 pN. We found that the yield strength of αIIbβ3-fibrinogen interactions was independent of the rate at which force was applied over a range of 160 to 16,000 pN/s, but the frequency of fibrinogen binding to αIIbβ3 during repeated contacts correlated strongly with the duration of contact between the αIIbβ3- and fibrinogen-coated surfaces. The observed rupture forces could be segregated into small (20–40 pN), intermediate (40–60 pN), and high (>60 pN) force regimes, each of which displayed unique kinetic behavior. Thus, the contact time needed to reach half maximal fibrinogen binding at a constant loading rate of 1,600 pN/s was ~ 1 ms for the 20–40 pN, 20 ms for the 40–60 pN, and 70 ms for the >60 pN force regimes. Increasing the surface density of immobilized fibrinogen changed the probability of αIIbβ3 binding to fibrinogen >3.5-fold, but did not affect the yield strength profile, implying that these measurements represent interactions between individual molecules. Each force regime also differed in its susceptibility to the inhibitory effect of αIIbβ3 antagonists and to the activating effect of Mn2+. Thus, the low molecular weight αIIbβ3 antagonists H12 peptide, tirofiban, and RGDS were most effective in inhibiting weak interactions, whereas the monoclonal antibodies A2A9 and abciximab were most effective in inhibiting the stronger ones. In the presence of Mn2+, the strongest component of the yield force distribution increased more than 2.5-fold while the moderate force component increased only 1.5-fold. In addition, rupture forces in the range of 60 to 150 pN disappeared gradually when αIIbβ3 preparations were stored at 4°C for 7 d, suggesting that the αIIbβ3 conformation corresponding to these rupture forces is labile. Taken together, these data suggest that fibrinogen binding to αIIbβ3 is a complex, time-dependent multi-step process during which the strength of the bond between αIIbβ3 and fibrinogen appears to progressively increase.
A Critical Role for Membrane Sulfatide in Platelet Aggregation.
Authors: Guchhait Prasenjit , Shrimpton Corie , Honke Kochi , Thiagarajan Perumal , Lopez Jose A. 
Published: October 2019

Abstract
Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.
PIP5Kγ-Null Mutation Induces Cytoskeletal Changes within Megakaryocytes.
Authors: Wang Yanfeng , Lian Lurong , Bach Tami L. , Chen Xinsheng , Chen Qing-Min , Abrams Charles S. 
Published: October 2019

Abstract
All eukaryotic cells contain the phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2) that serves multiple roles in signaling cascades critical for actin dynamics. Phosphatidylinositol-4-phosphate 5 kinase (PIP5K) catalyzes the synthesis of PIP2 by phosphorylating PI4P. Although the classical PIP5K isoforms (α, β, and γ) generate the same phospholipid product, the isoforms have different primary structures, are expressed at different levels in various tissues, and localize in different intracellular compartments. Therefore, it is likely that the functions of these isoforms are different as well. PIP5Kγ is unique because it associates directly with talin and consequently localizes in focal adhesion. Given the role of PIP2 in actin dynamics, we generated a murine line containing a null mutation in the PIP5Kγ gene. We found that the targeted disruption of PIP5Kγ results in early prenatal mortality that is associated with pleotropic effects involving the cardiovascular, neurologic, and hematopoietic systems. This early lethality prevented studies of hematopoietic cells derived from the bone marrow or the liver. However, we were able to analyze yolk sac progenitor cells that were treated with thrombopoietin ex vivo, differentiating them into megakaryocytes. After 5 days in culture, many of the non-adherent yolk sac progenitor cells were multinucleated, and approximately 70% expressed CD41 (αIIb), a marker of the megakaryocyte lineage. We then examined their cytoskeletal content and dynamics. Absence of PIP5Kγ had no effect on the quantity of F-actin in unstimulated or thrombin-stimulated cells that were adherent to fibrinogen or were maintained in suspension in the absence of stimulation. Next, we examined membrane dynamics during cell adhesion in real time using spinning disk video confocal microscopy. Wild type megakaryocytes actively formed and contracted lamellipodia and rapidly spread upon the fibrinogen matrix. In contrast, PIP5Kγ-null megakaryocytes continuously extended and retracted membrane blebs, rather than lamellipodia, but eventually spread as much as wild type cells. This observation is consistent with the previous suggestion that PIP2 contributes to the stable association of the membrane with the cytoskeleton. It is also noteworthy that a similar phenotype has been described in cells with defective anchoring of the membrane to the cytoskeleton because of a lack of filamin. Accordingly, our data are consistent with the hypothesis that PIP5Kγ generates compartmentalized pools of PIP2 that contribute to the association of the membrane with the cytoskeleton.
Manipulation of the Notch Pathway by γ-Secretase Inhibitors as a Novel Therapeutic Approach in Multiple Myeloma.
Authors: Jundt Franziska , Acikgoez Oezlem , Preissner Robert , Dorken Bernd 
Published: October 2019

Abstract
Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells. Thereby they control cell fate decisions and survival. We recently demonstrated that Notch has a pathogenetic role in multiple myeloma (MM), where tight interactions between neoplastic plasma cells and their microenvironment are essential for tumor cell growth (Blood.2004; 103:3511–3515). Our data provided evidence that Notch1 and Notch2 were highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts showed low to undetectable levels of Notch. Furthermore, our functional data indicated that activating the Notch pathway in MM cells by the Notch ligand Jagged1 potently induced tumor cell growth and suggested that these interactions contribute to myelomagenesis in vivo. In this study, we blocked Notch by novel γ-secretase inhibitors in cultured MM cells. γ-secretase catalyzes the release of the intracellular domain of Notch that subsequently translocates to the nucleus to activate expression of downstream target genes. Inhibition of γ-secretase activity is currently investigated as a therapeutic strategy in Alzheimer’s disease, because γ-secretase similarly cleaves amyloid precursor proteins to release Aβ peptides, accumulation of which is causally related to Alzheimer’s disease. To identify novel γ-secretase inhibitors that might block Notch in MM cells, we analyzed two million compounds by 3D in silico screening. Thereby structurally known inhibitors were compared with compounds from data banks. Novel structurally related compounds (27) were tested by in vitro assays. Specific inhibition of Notch signaling was controlled by RT-PCR of the down-regulated Notch target gene Hes-1 in MM cells. In addition, tumor cell proliferation of MM cells was efficiently blocked by 3 out of 27 newly identified inhibitors in a dose-dependent manner. Currently, we investigate whether these compounds also have potent anti-tumor activity against MM cells in mouse models in vivo. If so, interruption of Notch signaling by newly identified inhibitors might be a novel therapeutic principle to control the proliferation capacity of MM.
Pococurante (Poc): An ENU-Induced Missense Mutation of MyD88 with Selective Effects on TLR Signal Transduction.
Authors: Jiang Zhengfan , Li Chenglong , Shamel Louis , Olson Arthur , Beutler Bruce 
Published: October 2019

Abstract
Toll-like receptors (TLRs) are key sensors of the innate immune system, and individual TLRs respond to specific molecules derived from microbes. MyD88 is a Toll/Interleukin-1/Resistance (TIR) domain-containing adaptor protein required for signaling by all TLRs except TLR3. While the structural basis of association between MyD88 and TIR-domain receptors is obscure, MyD88-deficient mice show no responses to bacterial flagellin, peptidoglycan (PGN), lipoteichoic acid (LTA), bacterial lipopeptides such as PAM2CSK4, PAM3CSK4 and R- or S-MALP-2, DNA bearing unmethylated CpG dinucleotides (CpG DNA), or Resiquimod (RSQ). Using germline ENU mutagenesis, we have produced a large number of phenotypic variants that have abnormal TLR signaling. We now report the identification of a new mutation called Pococurante (Poc), originally detected in screening because macrophages from this mouse showed no response to the tri-acylated lipopeptide PAM3CSK4, the di-acylated lipopeptide S-MALP-2, LTA, CpG DNA, RSQ, and a markedly reduced response to LPS: the ligands for TLRs 2/1, 2/6, 9, 7 and 4 respectively. They also had no response to interleukin-1, a cytokine that signals by way of a MyD88-dependent TIR domain receptor. However, Poc mice showed a normal response to PGN, as well as R-MALP-2 and PAM2CSK4 lipopeptides. The latter three ligands are sensed in a TLR2-dependent, MyD88-dependent fashion. The Poc phenotype was ascribed to a point mutation of MyD88 affecting a surface residue (I179N). Because the mutation is discriminatory, permitting MyD88 to carry a signal from some TIR domain receptors but not others, we infer that it resides at the receptor:adaptor signaling interface. A new model of TIR receptor:adaptor interaction is proposed on the basis of docking studies that take account of the Poc phenotype, made using the protein-protein docking program SURFDOCK. We note that S-MALP-2 is dependent upon TLR2/6 heterodimers, while PAM3CSK4 sensing depends upon TLR2/1 heterodimers. Since the Poc mutation forbids detection of both these ligands while it allows detection of PAM2CSK4 and R-MALP-2, it may be inferred that TLR2 signal transduction entails greater structural diversity than was previously supposed. The involvement of TLR2 homodimers, or the incorporation of subunits yet unknown into the receptor complex, cannot be excluded.
Phenotypic Description of Familial Chronic Myeloproliferative Disorders.
Authors: Najman Albert , Chaumarel Isabelle , Bellann-Chantelot Christine , Barbu Veronique , Labopin Myriam , Rain Jean D. , Casadevall Nicole , Varet Bruno , Bauters Francis , Thomas Gilles 
Published: October 2019

Abstract
Chronic myeloproliferative disorders (CMPD) are rare diseases of the bone marrow characterised by a clonal proliferation of one or several myeloid lineages. Familial clustering are regularly reported suggesting a role for inherited factors. To adress this issue, we collected with the help of a large network of haematologists clinical data and blood samples of 78 families defined by the presence of at least two affected subjects with one of the four CMPD: polycythemia vera, essential thombocythemia, chronic myeloid leukemia and agnogenic myeloid metaplasia. A total of 176 affected subjects including 83 polycythemia vera, 70 essential thrombocythemia, 12 chronic myeloid leukemia and 11 agnogenic myeloid metaplasia were recruited. 449 asymptomatic relatives, mainly first-degrees, were collected and among them 11%were carriers of endogenous erythroid colonies with normal blood counts. Phenotypic spectrum within families was either homogenous (46/78) or heterogeneous (32/78) and the main association (25/32) was polycythemia vera and essential thrombocythemia. A few cases of changes in disease phenotype was also observed during the course of CMPD. Clinical and haematological data of affected subjects at diagnosis and during the course of the disease were similar to sporadic CMPD. However, a marked anticipation of approximatively 20 years/generation at onset age was observed. No excess of carcinomas was noted either in affected subjects or relatives. An excess of acute leukemias, however, was noted in relatives. We did not show evidence of known environmental factors such as exposition to ionic radiations or chemical solvents. Familial cases were restricted to a single generation in 33 families. Occurence was vertical in 45 families involving 2 generations in 36 and 3 generations in 9. In those familial cases consistent with a dominant inheritance, we exluded molecular abnormalities of VHL and EpoR genes involved in particular forms of familial and congenital polycythemia. In conclusion, the analysis of these 78 familial CMPD cases highlights that (i) the observation of mixed phenotypes in a large proportion is consistent with the theory that MPD arise from clonal expansion of a pluripotent hematopoietic precursor cell (ii) no environmental factor is clearly involved in the onset of CMPD and (iii) the observation of familial aggregations and the low incidence of the pathology suggest the implication of genetic predisposition factors in the occurence of myeloproliferative disorders. This large collection of multiplex families enables us to initiate genome-wide linkage analysis.
Role of Cell Adhesion in the Maintenance of Hematopoietic Stem Cells in the Bone Marrow Niche.
Authors: Arai Fumio , Hirao Atsushi , Hosokawa Kentaroh , Suda Toshio 
Published: October 2019

Abstract
Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis. We have demonstrated that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and anti-apoptotic, transplantable and comprise a side-population (SP) of HSCs, which contact closely to angiopoietin-1, a ligand for Tie2, expressing osteoblasts (OBs) in the BM niche. Tie2 and Ang-1 are part of a key signaling interaction between HSCs and niche cells. This signaling pathway regulates the feature of HSCs in the BM niche. The interaction of Tie2 with Ang-1 in vitro is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhanced the ability of HSCs to become quiescent and also induced their adhesion to bone surface, resulting protection of HSC compartment from stresses which suppress hematopoiesis (Arai et al., Cell, 2004). In this study, we focused on the role of cell adhesion molecule on the maintenance of HSCs in BM niche. A previous paper reported that both quiescent HSCs and some of osteoblasts express N-cadherin. We found that Tie2+ HSC expressed N- and VE-cadherin, and OBs expressed N-, P-, and OB-cadherin. This suggests that an adherens junction between HSCs and OBs created via N-cadherin may contribute to HSC maintenance. Furthermore, N-cadherin overexpressing OP9 stromal cells (OP9/N-cadherin) maintained LTC-IC more than control OP9 cells did. Overexpression of N-cadherin in HSCs also maintained colony formation. In addition, in the presence of Ang-1 in coculture of OP9/N-cadherin and HSCs facilitated the maintenance of CFU-C and HPP-CFC formation after long-term culture. Furthermore, Ang-1 treatment upregulated the expression of N-cadherin in Tie2+ HSCs as well as β1-integrin. It suggests that Tie2/Ang-1 signaling enhanced cell-cell adhesion between HSCs and OBs.
Altogether, these observations led us to the novel model that the localization of HSCs on bone surface is regulated by stem cell specific adhesion molecules such as N-cadherin. Once the HSCs adjacently localize to OBs, Ang-1 produced by OBs may activate Tie2 on the HSCs and promote tight adhesion of HSCs in the niche, resulting in quiescence and enhanced survival of HSCs.
Phenotypic and Functional Characterization of Smooth Muscle-Like Cells Derived from Multipotent Adult Progenitor Cells (MAPCs).
Authors: Ross Jeffrey J. , Keirstead Susan A. , Felton Mike , Tranquillo Robert T. , Verfaillie Catherine M. 
Published: October 2019

Abstract
Smooth muscle cells (SMC) play a key role in vascular development and morphogenesis of blood vessels defined by a noncontractile, or synthetic SMC phenotype. Mature SMCs respond to vascular injury/repair by phenotypic modulation to a synthetic state, and hence are also involved in various clinical disorders including atherosclerosis and cancer. We have induced relatively homogenous differentiation of SMC-like cells from multipotent adult progenitor cells (MAPC).
MAPCs have been isolated from murine, rat and human bone marrow. MAPCs can differentiate into mesodermal, neuroectodermal, and endodermal cell types. Here we report that serum free medium supplemented with PDGF-BB and TGF-b1 allows differentiation of rat MAPCs to a population of cells expressing smooth muscle specific markers, as determined by Q-RT-PCR (a-SMC actin, calponin, SM-22a, myocardin, and smooth muscle heavy chain myosin),and immunohistochemistry (a-SMC actin staining in >90% cells and calponin staining in ~50% of cells, co-localization localized with defined stress fibers). Such SMCs can subsequently be passed for at least 15 passages (26 doublings) in the presence of FCS and PDGF-BB and TGF-b1, without loss of phenotypic characteristics. RT-PCR analysis for genes of other mesodermal lineages, including cardiac muscle and endothelium expressed genes (Nkx, Mef-2D, PCAM, and vWF) was negative.
To characterize the function of the SMC-like cell population, calcium handling and in-vitro remodeling properties were studied. Passage 4 SMC-like cells responded to endothelin-1, norepinepherine, oxytocin, arg-vasopressin, and bradykinin with an increase in intracellular calcium ion concentration, consistent with a smooth muscle phenotype. To assess the remodeling potential, passage 4 cells were entrapped into fibrin matrix hemispheres as previously described for neonatal rat SMC. After 5 weeks in culture, hydroxyproline assays quantified total collagen as (86.8 +/− 9.3 ug) approximately 50% the amount reported using neonatal SMCs. In addition, the remodeled hemispheres demonstrated defined mechanical properties (tensile strength= 0.20 MPa, modulus = 0.65 MPa), similar to what we have previously observed when neonatal rat SMCs are entrapped in fibrin disks. Masson Trichrome staining demonstrates areas of tissue organization characterized by fibrils and collagen production. While further characterization of this population is needed to determine their developmental plasticity, the data is suggestive of a vascular SMC-like cell population derived from MAPCs that demonstrates functional smooth muscle characteristics and could be used to tissue engineer blood vessels.