bluuebunny/crossref_metadata_2025_split huggingface dataset:
Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy
Authors: Ginsel LA , Onderwater JJ , Fransen JA , Verhoeven AJ , Roos D 
Published: October 2019

Abstract
Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22- phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.
Sickle erythrocytes inhibit human endothelial cell DNA synthesis
Authors: Weinstein R , Zhou MA , Bartlett-Pandite A , Wenc K 
Published: October 2019

Abstract
Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling. These results indicate that interaction between sickle erythrocytes and the endothelium may result in altered endothelial function. This altered endothelial function may contribute to the development of vascular occlusive phenomena in patients with sickle cell anemia.
A frameshift mutation leading to type 1 antithrombin deficiency and thrombosis
Authors: Olds RJ , Lane DA , Finazzi G , Barbui T , Thein SL 
Published: October 2019

Abstract
Type 1 antithrombin III (ATIII) deficiency, which is the commonest form of inherited ATIII defect, is characterized by a quantitative reduction in both immunologically and functionally detectable protein. This condition is associated with a high incidence of thromboembolic disorder. Previous investigations have shown that the ATIII genes in the majority of cases are grossly intact, but the precise underlying molecular defects remain unknown. We have investigated the molecular basis of a type 1 ATIII deficiency in an Italian kindred by enzymatic amplification of the ATIII gene sequences in affected family members and direct sequencing of the amplified genomic DNA. A novel mutation, the deletion of a single T in the second position of codon 119, was identified in each of the affected individuals. The resulting frameshift leads to a premature termination in codon 126, effectively resulting in a null allele.
Different effect of benzylacyclouridine on the toxic and therapeutic effects of azidothymidine in mice
Authors: Falcone A , Darnowski JW , Ruprecht RM , Chu SH , Brunetti I , Calabresi P 
Published: October 2019

Abstract
It has been reported that in vitro uridine (Urd) can reverse azidothymidine (AZT) cytotoxicity without decreasing anti-human immunodeficiency virus (HIV) activity. Our studies in mice have shown that daily oral doses of benzylacyclouridine (BAU), an inhibitor of Urd breakdown, also reduces AZT hematologic toxicity, presumably by elevating the plasma concentration of Urd. We now extend these murine studies and report the effect of various doses of exogenous Urd, various doses of BAU, or the combination of BAU and Urd, administered daily, on AZT-induced toxicity. In mice receiving concomitant AZT, daily doses of Urd of 1,000 to 2,000 mg/kg increase peripheral reticulocytes and slightly reduce AZT-induced hematologic toxicity. However, the range of effective doses is narrow, and higher doses of Urd (greater than 3,000 mg/kg/d) significantly enhance hematologic toxicity. At its most effective dose, (2,000 mg/kg/d), Urd produces 28% mortality. In contrast, BAU doses up to 300 mg/kg/d reduced AZT-related hematologic toxicity in a dose-dependent manner without mortality. Higher daily doses of BAU and the combination of BAU with low doses of Urd were not more effective. Studies conducted in mice infected with the Rauscher murine leukemia virus (RLV) indicate that BAU does not impair the antiretroviral effect of AZT when administered at doses that reduce AZT-induced anemia and leukopenia. These findings may be significant for the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.
Turnover of *I-protein C inhibitor and *I-alpha 1-antitrypsin and their complexes with activated protein C
Authors: Laurell M , Stenflo J , Carlson TH 
Published: October 2019

Abstract
The rates of clearance and catabolism of human protein C inhibitor (PCI) and human alpha 1-antitrypsin (alpha 1-AT) and their complexes with human activated protein C (APC) were studied in the rabbit. The radioiodinated-free inhibitors had biologic half-lives of 23.4 and 62.1 hours, respectively, while the corresponding *I-labeled activated- protein C complexes were cleared with half-lives of 19.6 +/- 3.1 and 72.2 +/- 6.1 minutes. Complex clearances were linked to their catabolism as shown by a correlation between clearance and the appearance of free radioiodine in the plasma. Thus, the difference in the rates of catabolism would result in a fivefold greater amount of alpha 1-AT-APC complex than PCI-APC complex 1 hour after the formation of equal amounts of these in vivo. These results lead to the conclusion that the relative contribution of PCI and alpha 1-AT to the physiologic inhibition of APC cannot be determined only from the rates of the formation of these complexes in vitro, or from measurement of their levels in plasma. The APC-PCI complex is unstable as compared with the APC-alpha 1-AT complex, compounding the problem of estimating rates of complex formation from their levels in plasma.
Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937
Authors: Lindmark A , Persson AM , Olsson I 
Published: October 2019

Abstract
The processing of the neutral proteases cathepsin G and neutrophil elastase, normally synthesized in myeloid precursor cells and stored in azurophil granules, were investigated by biosynthetic labeling with 14C- leucine of the monoblastic cell line U-937. The proteases were precipitated with specific antibodies and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. The transfer to lysosomes of newly synthesized proteases was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenates in a Percoll gradient. The presence of a closely spaced polypeptide band-doublet at intermediate gradient density suggested cleavage of the specific aminoterminal pro dipeptide extension before storage in lysosomes. The molecular heterogeneity observed for cathepsin G and neutrophil elastase seemed to be due to modifications occurring after sorting into lysosomes, most likely because of C-terminal processing. Modifications of the secreted enzymes were not detectable by SDS-PAGE. In contrast to other lysosomal enzymes, no phosphorylation was demonstrated. Newly synthesized cathepsin G and neutrophil elastase rapidly became resistant to endoglycosidase H, indicating transport through the medial and trans cisternae of the Golgi complex and conversion to “complex” oligosaccharide side chains. This conversion was inhibited by an agent swainsonine, but translocation from the Golgi complex and secretion were unaffected. The processing described may play a role in activation of the proteases.
Transforming growth factor beta downregulates interleukin-1 (IL-1)- induced IL-6 production by human monocytes
Authors: Musso T , Espinoza-Delgado I , Pulkki K , Gusella GL , Longo DL , Varesio L 
Published: October 2019

Abstract
We investigated the effects of transforming growth factor beta (TGF beta) on the induction by interleukin-1 beta (IL-1 beta) of IL-6 in human monocytes. We found that IL-1 beta induced IL-6 messenger RNA expression in elutriated monocytes and IL-6 secretion in the supernatant. TGF beta did not induce IL-6. In contrast, TGF beta added to the culture inhibited, in a dose-dependent manner, the induction of IL-6 by IL-1 at the level of messenger RNA and bioactivity. These results show that IL-1 beta is able to stimulate IL-6 production by monocytes, TGF beta, by inhibiting this effect, may play an important role in regulating the IL-1-mediated components of the inflammatory response.
Sulfation of von Willebrand factor
Authors: Carew JA , Browning PJ , Lynch DC 
Published: October 2019

Abstract
von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated [35S]-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate- sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.
Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties
Authors: Grinnell BW , Walls JD , Marks C , Glasebrook AL , Berg DT , Yan SB , Bang NU 
Published: October 2019

Abstract
Human protein S (HPS), a regulator of hemostasis, is a vitamin K- dependent plasma protein with potential clinical utility. We have obtained high-level expression of the cDNA for HPS in two mammalian cell lines. Both cell lines secreted single chain recombinant HPS (rHPS) in serum-free medium as determined by Western blot analysis. The ability of the rHPS from both cell lines to act as a cofactor for human protein C (HPC) was determined; the rHPS secreted from the human 293 cell line had an activity six times that of the rHPS from the AV12–664 Syrian hamster cell line. Furthermore, the relative specific cofactor activity of rHPS from the 293 cell line was actually 2.5-fold higher than that of single-chain human plasma-derived HPS. Essentially all of the rHPS secreted from the 293 cell line exhibited a calcium-dependent elution profile on anion exchange chromatography, whereas only 25% to 35% of the hamster cell-derived rHPS exhibited this profile. However, the calcium-eluted rHPS from the AV12 cell line had a high specific cofactor activity, equivalent to that of the 293-derived rHPS. A NaCl- elutable rHPS fraction (calcium nondependent) was isolated from the recombinant AV12–664 cell line, further purified, and found to have reduced activity, only 40% that of the calcium-dependent rHPS. The only observable difference in the calcium-dependent and nondependent rHPS molecules was in the content of gamma-carboxyglutamic acid (Gla); the calcium-dependent material contained approximately 10 mol Gla/mol protein whereas the calcium-nondependent material contained only approximately 8 mol Gla/mol of protein. In addition, the calcium- nondependent rHPS had reduced ability to interact with phospholipid vesicles as evidenced by an eightfold increase in the apparent kd. Our data demonstrate the isolation of rHPS with high specific activity, and show that a reduction in as few as two Gla residues dramatically decreases its functional cofactor activity for HPC, due to a reduction in ability to interact with the phospholipid bilayer.
Time-dependent alterations in the deformability of human neutrophils in response to chemotactic activation
Authors: Frank RS 
Published: October 2019

Abstract
Transit times of individual human neutrophils through single capillary- sized pores were measured to determine the time-dependent changes in the mechanical behavior of the cells during activation by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (FMLP) and in response to cytochalasin B (CTB) and colchicine. FMLP elicited a two- phase response consisting of a rapid increase in cell stiffness, which peaked between 2 and 3 minutes, followed by a partial recovery of deformability to a level significantly above that of control after 5 minutes. The mechanical changes closely followed changes in F-actin content, although the peak in cell stiffness appeared to lag the F- actin response. Treatment with 3 mumols/L CTB produced a transient decrease in cell rigidity followed by a return to control level in 10 minutes, whereas treatment with 30 mumols/L CTB resulted in a sustained decrease in cell transit times to a level 60% of control. Addition of 3 mumols/L CTB to cells prestimulated with FMLP produced a rapid (1 to 2 minutes) cessation of changes in cellular deformability produced by the FMLP. Colchicine treatment did not decrease cell rigidity, but produced a delayed increase in F-actin content accompanied by increased stiffness of the cells. These results implicate actin as the major determinant of the mechanical behavior of the neutrophil, as measured by whole cell deformability tests. The significant changes in cell deformability that occur in response to fractional changes in F-actin content suggest that changes in the structure of the actin network occur during these processes.
Human serum megakaryocyte colony-stimulating activity appears to be distinct from interleukin-3, granulocyte-macrophage colony-stimulating factor, and lymphocyte-conditioned medium
Authors: Mazur EM , Cohen JL , Newton J , Sohl P , Narendran A , Gesner TG , Mufson RA 
Published: October 2019

Abstract
Sera from patients with bone marrow megakaryocyte aplasia are a rich source of megakaryocyte colony-stimulating activity (Meg-CSA). Other biologic materials exhibiting Meg-CSA include phytohemagglutinin- stimulated human lymphocyte-conditioned medium (PHA-LCM), recombinant interleukin-3 (IL-3), and recombinant granulocyte macrophage colony- stimulating factor (GM-CSF). Neutralizing antisera to both recombinant IL-3 and GM-CSF were used to evaluate the relationship among these sources of Meg-CSA. Varying dilutions of IL-3 and GM-CSF antisera were tested in plasma clot cultures of normal human peripheral blood megakaryocyte progenitors optimally stimulated by either IL-3 (1 U/mL), GM-CSF (1 U/mL), PHA-LCM (2.5% to 5% vol/vol), or aplastic human serum (10% vol/vol). IL-3 antiserum at dilutions up to 1/2,000 totally abrogated megakaryocyte colony growth stimulated by IL-3. A 1/500 dilution of GM-CSF antiserum completely eliminated GM-CSF-induced megakaryocyte colony development. A combination of anti-IL-3 and anti- GM-CSF, each at a 1/500 dilution, inhibited all megakaryocyte colony growth stimulated by optimal concentrations of IL-3 and GM-CSF together. There was no neutralizing crossreactivity between the IL-3 and GM-CSF antisera. At maximally neutralizing concentrations, IL-3 antiserum inhibited 66% of the megakaryocyte colony growth stimulated by PHA-LCM. Residual megakaryocyte colony growth was eliminated by the addition of a 1/500 dilution of anti-GM-CSF.
In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice
Authors: Rennick DM , Thompson-Snipes L , Coffman RL , Seymour BW , Jackson JD , Hudak S 
Published: October 2019

Abstract
Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5- responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.
Enhanced stimulation of human bone marrow macrophage colony formation in vitro by recombinant human macrophage colony-stimulating factor in agarose medium and at low oxygen tension
Authors: Broxmeyer HE , Cooper S , Lu L , Miller ME , Langefeld CD , Ralph P 
Published: October 2019

Abstract
Recombinant (r) and natural human (h) macrophage colony-stimulating factor (M-CSF, CSF-1) have been considered poor stimulators of macrophage progenitor cells present in human marrow, although they are potent stimulators of these cells in mouse marrow. We compared the growth characteristics of rhM-CSF-responsive human macrophage progenitor cells placed in semisolid agarose or agar culture medium and incubated for 14 days at ambient (approximately 20%) or lowered (5%) O2 tension. By itself, rhM-CSF was found to be a good stimulator of macrophage colony formation by human bone marrow cells cultured in agarose but not in agar; this growth was enhanced by incubation at 5% O2. Maximal numbers (up to 115/10(5) nonadherent low density cells plated) of macrophage colonies (50 to greater than 500 cells per colony) were stimulated by 500 to 1,000 units rhM-CSF/mL, with 1/2 maximal numbers stimulated by 250 to 500 units/mL. With agarose as the support medium, rhM-CSF was two- to fourfold more active on mouse than on human macrophage colony formation, in contrast to previous reports of 10- to 100-fold greater activity when agar was used as the support medium. Using nonadherent low density T lymphocyte-depleted human bone marrow cells growing in agarose at 5% O2, greater than additive effects on colony formation were observed when 31 to 500 units rhM-CSF were used in combination with either 10 ng rh interleukin-1 alpha (IL-1 alpha), 20, or 200 units rh granulocyte-macrophage (GM)-CSF or rhG-CSF. The agarose assay system should be useful for evaluating factors regulating the proliferation of human macrophage progenitor cells in vitro and during clinical trials with rhM-CSF.
Consecutive enzyme cascades: complement activation at the cell surface triggers increased tissue factor activity
Authors: Carson SD , Johnson DR 
Published: October 2019

Abstract
Complement activation at the cell surface initiates cell damage through a series of reactions occurring at the cell membrane and, after assembly of the terminal membrane attack complex, produces leakage of cytoplasmic contents from the cell. It has been documented that chemical or physical damage to cell membranes can cause a rapid increase in the expression of tissue factor procoagulant activity. In this study, antibody-mediated complement activation at the cell surface resulted in increased tissue factor activity, which correlated with cytolysis, as measured by 51-chromium release. Therefore, complement fixation on the cell surface can have a direct and immediate stimulatory effect on the coagulation cascade at the point of its initiation, with formation of a fibrin clot requiring only three consecutive proteolytic reactions after immunologically mediated cell damage.