bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1
Authors: Li XK , Lijnen HR , Nelles L , Van Hoef B , Stassen JM , Collen D 
Published: October 2019

Abstract
A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296- G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296- G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt- PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS).
Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease
Authors: Tycko B , Ritz J , Sallan S , Sklar J 
Published: October 2019

Abstract
A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.
Clinical and immunologic effects of prolonged infusion of low-dose recombinant interleukin-2 after autologous and T-cell-depleted allogeneic bone marrow transplantation
Authors: Soiffer RJ , Murray C , Cochran K , Cameron C , Wang E , Schow PW , Daley JF , Ritz J 
Published: October 2019

Abstract
Bone marrow transplantation (BMT) can produce prolonged clinical remission in some patients with hematologic malignancies. Unfortunately, disease relapse may occur despite BMT. Studies in animal models and clinical experience have provided evidence that immunologic factors play an important role in preventing relapse post-BMT. To stimulate immunologic activity in patients post-BMT, we administered prolonged uninterrupted continuous infusions of low-dose recombinant interleukin-2 (rIL-2). Thirteen marrow recipients (seven autologous BMT, six CD6 T-depleted allogeneic BMT) received rIL-2 at a dose of 2 x 10(5) U/m2/d for a scheduled period of 90 days. rIL-2 was administered through a Hickman catheter with a portable pump beginning a median of 85 days after BMT. Toxicity was minimal and all treatment could be undertaken in the outpatient setting. No patient developed any signs of graft-versus-host disease, hypotension, or pulmonary capillary leak syndrome. Treatment did not affect the absolute neutrophil count or hemoglobin level, but eosinophils increased substantially in most patients. Platelet counts decreased by 20% in 10 of 13 individuals within 2 weeks, but stabilized thereafter. Despite the low dose of rIL- 2 administered, significant immunologic changes were noted. Specifically, all 13 patients experienced a marked increase (fivefold to 40-fold) in natural killer (NK) cell number. Phenotypic characterization showed that the majority of NK cells were CD56bright+ CD16+ CD3-. In contrast, a minor increase in T-cell number was noted in only 4 of 13 patients. Low-dose rIL-2 treatment resulted in augmentation of in vitro cytotoxicity against K562 and COLO tumor targets. This cytotoxic activity could be dramatically enhanced by incubation with additional rIL-2 in vitro. The immunologic effects of rIL-2 treatment were similar in both autologous and allogeneic marrow recipients. Our data suggest that prolonged infusion of rIL-2 at low doses is safe and can selectively increase NK cell number and activity after BMT. Further studies to assess the impact these changes may have on disease relapse post-BMT will be undertaken.
Selection of histocompatible apheresis platelet donors by cross- matching random donor platelet concentrates
Authors: O'Connell BA , Lee EJ , Rothko K , Hussein MA , Schiffer CA 
Published: October 2019

Abstract
It can be impossible to identify compatible platelet donors for alloimmunized patients whose HLA type cannot be determined or who have uncommon HLA types. We have previously shown that histocompatible donors can be rapidly identified by “mass screening” of platelet concentrates (PC), which are readily available in all blood banks, using a solid-phase adherence platelet cross-matching technique. Compatible PC were given to five alloimmunized patients with multispecific HLA antibodies refractory to random donor (RD) PC and selected single-donor platelet transfusions. After transfusions which produced satisfactory responses, we identified the original whole blood donors to serve as apheresis donors. Thus, the donors selected were compatible in vitro by cross-matching, and in vivo by transfusion. Only 3% to 13% of PC cross-matched for these alloimmunized patients were potentially compatible and it was necessary to screen large numbers (65 to 205 U) of PC per patient. Eighteen of 22 PC-selected transfusions produced satisfactory increments, allowing selection of 12 donors, all of whom were willing to undergo apheresis. Ten of 12 of these single- donor transfusions were successful; the two unsuccessful transfusions were infused 2 weeks after the initial PC cross-match and were still compatible with the original serum, but incompatible with more recent serum, demonstrating a change in antibody reactivity. The HLA types of the successful single donors selected by PC cross-matching differed widely from the patients' HLA types and, therefore, these donors would not have been selected by standard approaches using HLA typing. Cross- matching large numbers of RD PC for the identification of apheresis donors is helpful in the management of the alloimmunized patient and may be of particular utility for blood centers that do not have access to HLA-typed donor pools.
Transcription of the human colony-stimulating factor-1 receptor gene is regulated by separate tissue-specific promoters
Authors: Roberts WM , Shapiro LH , Ashmun RA , Look AT 
Published: October 2019

Abstract
Receptors for macrophage colony-stimulating factor (CSF-1R) are expressed not only by monocytes, macrophages, and their progenitors, but also by placental trophoblasts during fetal development. In monocytes, CSF-1R gene transcripts originate at multiple sites immediately upstream of the gene's coding sequences, whereas in placental cells the transcripts include an additional noncoding exon, located 26 kb upstream near the 3′ end of the B-type platelet-derived growth factor (PDGF) receptor gene. Physically distinct CSF-1R transcription origins suggest separate promoter usage by the two cell types. To identify regulatory elements of these promoters, we fused CSF- 1R genomic sequences to bacterial reporter genes and introduced the resulting constructs into human cell lines and mouse fibroblasts. A 775- bp genomic fragment containing CSF-1R placental transcription origins and adjacent upstream sequences mediated reporter gene expression in BeWo choriocarcinoma cells and mouse NIH-3T3 fibroblasts, but not in myeloid or lymphoid cells. By contrast, a 550-bp genomic fragment containing CSF-1R monocyte transcription origins and 5′ flanking sequences directed gene expression in U-937 human myeloid cells, but not in the other cell types. Thus, nucleotide sequences of fewer than 1,000-bp upstream of the two independent CSF-1R transcription origins contain the minimal promoter elements needed to program appropriate tissue-specific expression of reporter genes.
Immunovisualization of high (HK) and low (LK) molecular weight kininogens on isolated human neutrophils
Authors: Figueroa CD , Henderson LM , Kaufmann J , De La Cadena RA , Colman RW , Muller- Esterl W , Bhoola KD 
Published: October 2019

Abstract
An immunocytochemical study was performed to examine the cellular localization and the subcellular distribution of kininogens in human blood cells. Kininogens were visualised using the immunogold-silver staining method and confocal scanning laser microscopy. We confirmed the existence of high molecular weight kininogen in human neutrophils and describe for the first time the presence of low molecular weight kininogen on these cells. Both high and low molecular weight kininogens were restricted to the neutrophils where they localized as clusters of immunogold particles on the cell membrane. No labeling was observed intracellularly in organelles such as mitochondria, endoplasmic reticulum, and azurophilic or specific granules after permeabilization of the neutrophils with Triton X-100, a procedure that permitted the visualization of elastase in the azurophilic granules. Clusters of high molecular weight kininogen molecules attached to the neutrophil surface could serve as receptors for plasma kallikrein and/or be the source of substrate for a discrete and circumscribed formation of kinins that may in turn facilitate the local diapedesis of neutrophils and the transudation of plasma constituents during acute inflammation.
Effect of activin A on globin gene expression in purified human erythroid progenitors
Authors: Shao L , Frigon NL Jr , Young AL , Yu AL , Mathews LS , Vaughan J , Vale W , Yu J 
Published: October 2019

Abstract
The regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progenitors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all alpha, beta, and epsilon globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125I-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-beta. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s).
Mechanisms of amphipath-induced stomatocytosis in human erythrocytes
Authors: Schrier SL , Zachowski A , Devaux PF 
Published: October 2019

Abstract
We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin- labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration- dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet.
Membrane orientation of Rh(D) polypeptide and partial localization of its epitope-containing domain
Authors: Suyama K , Goldstein J 
Published: October 2019

Abstract
We have previously shown that the effects of various enzyme treatments on Rh antigen-containing polypeptides in situ could be monitored by an antibody preparation which recognizes only these polypeptides following Western blotting. We now have prepared antibodies that specifically react with either the N- or C-terminal ends of Rh-related proteins. Using all three, we have established that the C-terminus of Rh(D) polypeptide is at the cell surface, whereas its N-terminal domain is situated at the cytoplasmic side of the red blood cell membrane. Chymotrypsin digestion of ghosts derived from (-D-/-D-) cells that are devoid of Rh (C/c) and (E/e) antigens produces three major Rh(D)- related fragments: the 20-Kd fragment contains the molecule's C- terminal domain, the 17-Kd fragment its N-terminus, and the 13-Kd fragment neither. However, only the 17-Kd fragment forms an immune- complex with human polyclonal anti-D, indicating that it contains the Rh(D) antigenic domain. Other findings presented here provide further evidence for a unique folding of Rh(D) polypeptide within the cell membrane and suggest that Rh(C/c) and (E/e) polypeptides, when present, may form complexes with it.
Involvement of the putative hematopoietic transcription factor SCL in T- cell acute lymphoblastic leukemia
Authors: Aplan PD , Lombardi DP , Reaman GH , Sather HN , Hammond GD , Kirsch IR 
Published: October 2019

Abstract
The SCL gene, initially discovered at the site of a translocation breakpoint associated with the development of a stem cell leukemia, encodes a protein that contains the highly conserved basic helix-loop- helix (bHLH) motif found in a large array of eukaryotic transcription factors. Recently, we have described a nonrandom, site-specific SCL rearrangement in several T-cell acute lymphoblastic leukemia (ALL) cell lines that juxtaposes SCL with a distinct transcribed locus, SIL. The SIL/SCL rearrangement was found in leukemic blasts from 11 of 70 (16%) newly diagnosed T-cell ALL patients, a prevalence substantially higher than that of the t(11;14) translocation, which has previously been reported as the most frequent nonrandom chromosomal abnormality in T- cell ALL. We did not detect the SIL/SCL rearrangement in the leukemic blasts from 30 patients with B-cell precursor ALL, indicating that the rearrangement was specific for T-cell ALL. Analysis of RNA from these patients indicated that an SIL/SCL fusion mRNA was formed, joining SIL and SCL in a head-to-tail fashion. The fusion occurs in the 5′ untranslated region (UTR) of both genes, preserving the SCL coding region. The net result of this rearrangement is that SCL mRNA expression becomes regulated by the SIL promoter, leading to inappropriate SCL expression. The resultant inappropriate expression of this putative transcription factor may then contribute to leukemic transformation in T-cell ALL.
Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity
Authors: Blajchman MA , Fernandez-Rachubinski F , Sheffield WP , Austin RC , Schulman S 
Published: October 2019

Abstract
Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.
Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura
Authors: Fujisawa K , Tani P , O'Toole TE , Ginsberg MH , McMillan R 
Published: October 2019

Abstract
Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to antiplatelet autoantibodies, many of which are directed against platelet membrane glycoprotein (GP) IIb-IIIa or GPIb-IX. In a recent study, we described plasma autoantibodies from 13 selected ITP patients, which required the presence of the putative GPIIIa cytoplasmic region for antibody binding. Since this region may not be available for antibody binding under physiologic conditions, we evaluated the frequency of binding to this or other regions of GPIIb- IIIa by platelet-associated and plasma autoantibody from a group of chronic ITP patients. We studied platelet-associated autoantibodies in 27 patients and plasma antibodies in 21 patients; in 15 patients, both were studied. To determine if autoantibodies were directed to the cytoplasmic portion of GPIIIa or to another portion of the GPIIb-IIIa molecule, antibody eluted from patient platelets or plasma antibody was tested in an antigen capture assay for binding to GPIIb-IIIa obtained from Chinese hamster ovary (CHO) cells transfected with GPIIb and either intact GPIIIa or GPIIIa lacking the carboxy terminal 35 residues. Of the 21 plasma autoantibodies tested, 13 bound primarily to the carboxy terminus of GPIIIa and eight to other epitopes. Conversely, all 26 platelet-associated autoantibodies, including eight of the 13 with anti-carboxy terminus antibodies, bound to epitopes in other regions of GPIIb-IIIa. Comparison of the degree of antibody adsorption by intact or lysed platelets indicated that epitopes on the c-terminal region of GPIIIa are relatively inaccessible on the surface of intact washed platelets when compared with other epitopes. We conclude that the importance of plasma autoantibodies in chronic ITP patients should be interpreted cautiously, since their specificity may differ from that of antibodies bound to the platelet. Whether antibodies against the c- terminus of GPIIIa are of pathogenetic importance remains to be determined, although patients with these antibodies have particularly severe disease.