bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors
Authors: Carter RF , Abrams-Ogg AC , Dick JE , Kruth SA , Valli VE , Kamel-Reid S , Dube ID 
Published: October 2019

Abstract
Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.
Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells
Authors: Avraham H , Vannier E , Cowley S , Jiang SX , Chi S , Dinarello CA , Zsebo KM , Groopman JE 
Published: October 2019

Abstract
The kit ligand (KL), also termed stem cell factor (SCF), is a recently discovered hematopoietic growth factor that augments response of early progenitor cells to other growth factors and supports proliferation of continuous mast cell lines. Histological studies suggest that the receptor for SCF/KL, the c-kit proto-oncogene product, is present in bone marrow megakaryocytes. We studied the effects of SCF/KL on immortalized human megakaryocytic cell lines (CMK, CMK6, and CMK11–5) and on isolated human marrow megakaryocytes. Human SCF/KL alone or in combination with the hematopoietic growth factors, interleukin-3 (IL- 3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL- 6, stimulated proliferation of these megakaryocytic cell lines. SCF/KL treatment did not alter expression of gpIb, gpIIb/IIIa, LFA-1, ICAM-1, or GMP-140 in CMK cells. No effect on ploidy was observed. Furthermore, human SCF/KL induced expression of IL-1 alpha, IL-1 beta, IL-2, and IL- 6 in CMK cells. In a fibrin clot system, SCF/KL modestly potentiated megakaryocyte colony formation when added alone to cultures containing CD34+, DR+ bone marrow cells. Addition of SCF/KL with IL-3 or GM-CSF to these cultures resulted in a more marked marrow megakaryocytic cells. SCF/KL may directly affect megakaryocytopoiesis, as well as secondarily modulate hematopoiesis through induction of cytokines in target cells.
Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome
Authors: Bevers EM , Wiedmer T , Comfurius P , Shattil SJ , Weiss HJ , Zwaal RF , Sims PJ 
Published: October 2019

Abstract
The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.
A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage
Authors: Pittman DD , Millenson M , Marquette K , Bauer K , Kaufman RJ 
Published: October 2019

Abstract
Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site- directed DNA-mediated mutagenesis. Thrombin-activated human recombinant- derived factor VIII was isolated in an active form by passage over Mono- S fast protein liquid chromatography. The peak fractions had a specific activity of 60,000 U/mg. The subunit composition in the peak fraction contained the 50-Kd A1 domain from the heavy chain, the 73-Kd light chain fragment, and trace amounts of the 43-Kd A2 domain. The requirement of domain A2 for functional activity was shown in several ways. First, the addition of an inhibitory monoclonal antibody that recognizes domain A2 destroyed factor VIIIa activity. Second, addition of a Mono-S FPLC fraction that contained the A2 domain polypeptide back to the peak activity fraction increased activity of the factor VIIIa by 22-fold. The maximum specific activity achieved was 180,000 U/mg. Finally, expression of an A2 domain deletion mutant did not yield procoagulant activity, although the mutant was effectively secreted from the cell, exhibited appropriate heavy and light chain association, and was susceptible to thrombin cleavage. Cotransfection of this A2 domain deletion mutant with an A2 domain expression vector yielded a secreted complex and restored procoagulant activity in the conditioned medium. This result shows that the A2 domain can fold and assemble with A2-deleted factor VIII to yield a functional molecule. We conclude that the A2 domain is required for functional factor VIIIa activity and loss of activity in activated factor VIII may result from dissociation of A2 from the thrombin-activated heterotrimer.
Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ib alpha leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease
Authors: Miller JL , Lyle VA , Cunningham D 
Published: October 2019

Abstract
The primary sequences of the three individual glycoprotein (GP) chains, GPIb alpha, GPIb beta, and GPIX, comprising the normal platelet GPIb/IX receptor for von Willebrand factor (vWF) have recently been determined, opening the possibility for characterization of disorders of this receptor at the molecular level. The presence of a leucine tandem repeat in each of these chains is of particular interest, because such repeats may be involved in associations between polypeptide segments. We now report an autosomal dominant variant of Bernard-Soulier disease associated with the heterozygous substitution of phenylalanine for a highly conserved leucine residue within the GPIb alpha leucine tandem repeat. Affected individuals experienced a moderate bleeding tendency, thrombocytopenia, and an increased mean platelet volume. Platelet aggregation was decreased only in response to ristocetin or to asialo- vWF. The kd for 125I-vWF binding to patients platelets was significantly increased over control values at 0.5 mg/mL ristocetin, but was normal at 1.0 or 1.5 mg/mL ristocetin. While sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an essentially normal complement of all components of the GPIb/IX complex, a minor amount of a putative proteolytic fragment was identified that migrated faster than GPIb and was immunoreactive with polyclonal anti-GPIb alpha antibody, but not with a monoclonal antibody directed against the 45-Kd amino-terminal region of GPIb alpha. However, because the great majority of patient GPIb alpha comigrates with normal GPIb alpha, the major functional abnormalities of the patient platelets are most likely a consequence of the altered structure of the nonproteolyzed protein. Full concordance within the studied family between phenotypic expression and a heterozygous single nucleotide substitution in genomic DNA coding for a phenylalanine in place of the wild-type leucine at residue 57 of the mature GPIb alpha, absence of this substitution in 266 alleles from the normal population, and the lack of any other abnormality of patient DNA throughout the entire coding sequence for GPIb alpha provide strong support that this substitution may constitute a pathologic point mutation responsible for the observed phenotypic abnormalities. While the roles that leucine tandem repeats may normally play within the GPIb/IX complex are not yet known, the perturbation of such a repeat in GPIb alpha may impair interaction with other components of the complex and/or with the binding of vWF.
Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6
Authors: Natsuka S , Akira S , Nishio Y , Hashimoto S , Sugita T , Isshiki H , Kishimoto T 
Published: October 2019

Abstract
NF-IL6 was originally identified as a DNA binding protein regulating interleukin-1 (IL-1)-stimulated IL-6 expression. Direct cloning of NF- IL6 showed its homology with C/EBP, a hepatocyte- and adipocyte- specific transcription factor. This study showed that the expression of NF-IL6 messenger RNA (mRNA) increased markedly during the differentiation to a (mRNA) increased markedly during the differentiation to a macrophage lineage in mouse myeloid leukemia cells M1, human histiocytic leukemia cells U937, promyelocytic leukemia cells HL-60, and human peripheral monocytes. Particularly in HL-60 cells that undergo granulocyte or macrophage differentiation depending on inducers, NF-IL6 mRNA was specifically upregulated during macrophage differentiation but not granulocyte differentiation. It was also shown that the functional NF-IL6 protein increased during the differentiation of U937 cells. Furthermore, recombinant NF-IL6 was found to bind to the regulatory regions of the IL-1, tumor necrosis factor, granulocyte colony-stimulating factor, and lysozyme genes, which are expressed in mature macrophages. These results suggest that NF-IL6 may possibly be involved as an important transcription factor in the process of activation and/or differentiation of macrophages.
Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells
Authors: Pettersson M , Jernberg-Wiklund H , Larsson LG , Sundstrom C , Givol I , Tsujimoto Y , Nilsson K 
Published: October 2019

Abstract
The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the Ig loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bcl-2 locus were evident in only one of the myeloma cell lines, U-266–1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266– 1984), suggesting that bcl-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast to previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bcl- 2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells.
The mutation Arg (53)----Trp causes von Willebrand disease Normandy by abolishing binding to factor VIII. Studies with recombinant von Willebrand factor
Authors: Jorieux S , Tuley EA , Gaucher C , Mazurier C , Sadler JE 
Published: October 2019

Abstract
von Willebrand factor (vWF) and factor VIII (FVIII) circulate in plasma as a noncovalently linked protein complex. The FVIII/vWF interaction is required for the stabilization of procoagulant FVIII activity. Recently, we reported a new variant of von Willebrand disease (vWD) tentatively named “Normandy,” characterized by plasma vWF that appears to be structurally and functionally normal except that it does not bind FVIII. Three patients from one family were found to be homozygous for a C----T transition at codon 816 converting Arg 53 to Trp in the mature vWF subunit. To firmly establish a causal relationship between this missense mutation and vWD Normandy phenotype, we have characterized the corresponding recombinant mutant vWF(R53W). Expressed in COS-7 cells or CHO cell lines, normal vWF and vWF(R53W) were processed and formed multimers with equal efficiency. However, vWF(R53W) exhibited the same defect in FVIII binding as did plasma vWF from patients with vWD Normandy, confirming that this mutation is responsible for the vWD Normandy phenotype. These results illustrate the importance of Arg 53 of the mature vWF subunit for the binding of FVIII to vWF, and identify an amino acid residue within a disulfide loop not previously known to be involved in this interaction.
Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion
Authors: Grossbard ML , Freedman AS , Ritz J , Coral F , Goldmacher VS , Eliseo L , Spector N , Dear K , Lambert JM , Blattler WA 
Published: October 2019

Abstract
Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin “blocked ricin.” The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.
Blood flow to bone marrow during development of anemia or polycythemia in the rat
Authors: Iversen PO , Nicolaysen G , Benestad HB 
Published: October 2019

Abstract
We applied the radioactive microsphere method to follow the magnitude and time course (0 to 96 hours) of blood flow changes during development and recovery from anemia in awake rats. Blood flow was also monitored during a 96-hour period after polycythemia was induced (erythropoietin administered subcutaneously [SC]). The possible influence of innervation was also examined. After a blood loss of approximately 50% (hypovolemia), blood flow to the femoral marrow tripled within 12 hours and remained elevated for the entire 96-hour period. The relative increase in blood flow to the femoral bone was even greater. Similar findings were obtained in rats with phenylhydrazine (PHZ) hemolytic anemia (normovolemia). Denervation had no detectable effect on the increased blood flow to either marrow or bone. The augmented blood flow during hemolytic anemia was accompanied by a doubling of the oxygen consumption rate by the marrow, while the glucose uptake was not detectably altered. Erythropoietin supplements (3 x 1,000 IU/kg, SC, 6-hour intervals) increased blood flow to the marrow by approximately 25% after 48 hours, and at 72 hours the blood flow had reached a value twice that obtained under control conditions. These results indicate that blood flow to bone marrow is highly variable and hormonally and/or locally regulated. This may have practical consequences for marrow transplantation technology and for administration of drug therapy to patients with insufficient bone marrow hematopoiesis.
Identification and sequence analysis of the promoter for the leukocyte integrin beta-subunit (CD18): a retinoic acid-inducible gene
Authors: Agura ED , Howard M , Collins SJ 
Published: October 2019

Abstract
Leukocyte adhesion receptors (LFA-1; Mac-1; p150,95) are a family of heterodimeric cell-surface adhesion molecules expressed exclusively in granulocytes, lymphocytes, and macrophages. Expression of these proteins is under complex regulatory control, but to date promoters for these genes have not been identified. The CD18 gene codes for the common beta-subunit of the leukocyte adhesion receptors. Transcription of CD18 is highly tissue-specific, hormonally inducible (by retinoic acid [RA]), and coordinately regulated with leukocyte integrin alpha- chains. To identify the CD18 promoter, we screened a human genomic phage library with a human CD18 cDNA probe and obtained a clone that contains an exon coding for the 5′ untranslated region (UTR). Using rapid amplification of cDNA ends (RACE), RNAse protection, S1 nuclease, and primer extension assays, we demonstrated the existence of multiple transcription start sites clustered in a 45-nt region. We investigated the transcription-promoting activity of the genomic sequences 5′ to the CD18 gene by performing transient expression assays with a growth hormone reporter gene in various hematopoietic cell lines. The CD18 promoter was active in Jurkat cells, a lineage that normally expresses CD18 but was considerably less active in K562, an early erythroid line that does not normally express CD18. The genomic sequences upstream of the start site cluster lack CAAT and TATA boxes, but have two Sp1 binding sites and 10 T(G/C)AC(C/A) boxes, which may represent binding sites for RA receptors (RAR). These features distinguish the CD18 promoter from the promoters of other tissue-specific, hormone-inducible genes, and may be representative of leukocyte integrin promoters in general.
Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique
Authors: Britigan BE , Roeder TL , Shasby DM 
Published: October 2019

Abstract
Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA- stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy- radical generation.