scholarly journals A fast-acting elastase inhibitor in human monocytes.

1985 ◽  
Vol 162 (6) ◽  
pp. 2142-2155 ◽  
Author(s):  
E Remold-O'Donnell
Keyword(s):  
Proteinase Inhibitor ◽  
Sodium Dodecyl ◽  
Human Monocyte ◽  
Sulfate Solution ◽  
Cell Protein ◽  
Human Monocytes ◽  
High Concentration ◽  
Elastase Inhibitor ◽  
Nucleophilic Cleavage ◽  
Fast Acting

A proteinase inhibitor active against neutrophil and pancreatic elastase was detected in extracts of cultured human monocytes and the human monocyte-like cell line U937. This component forms a covalent complex with the active site of elastase; the complex is stable in boiling sodium dodecyl sulfate solution, and is susceptible to nucleophilic cleavage. The activity of the elastase inhibitor is not detected in extracts of freshly isolated monocytes, but becomes detectable when the monocytes are allowed to mature in culture, with maximum levels occurring at 5-7 d. The monocyte inhibitor is fast-acting; its reaction with 125I-labeled elastase is complete in less than 1 min at 37 degrees C. Analysis by electrophoresis and studies using a heteroantiserum to alpha 1-proteinase inhibitor demonstrated that the elastase inhibitor of monocytes/U937 cells is not identical to alpha 1-proteinase inhibitor, the major elastase inhibitor of blood plasma. The extent of conversion of 125I-elastase to the 125I-elastase-inhibitor complex is proportional to the amount of U937 extract or cultured monocyte extract, indicating that this reaction can serve to quantify the elastase inhibitor. The elastase inhibitor is an abundant component in mature monocytes, with greater than or equal to 1.5 X 10(6) molecules/cell (greater than or equal to 12 micrograms per 10(8) cells, greater than 0.1% of total cell protein). Its mol wt is estimated at 50,000. Thus, the monocyte inhibitor should be classified as a putative regulator of neutrophil (and monocyte) elastase activity at inflammatory sites. This designation is based on the properties of the molecule, including its high concentration in maturing monocytes, its affinity for elastase, and its fast reaction with this enzyme.

scholarly journals Radiation effects on cultured human monocytes and on monocyte-derived macrophages

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1402-1407 ◽  
Author(s):  
ES Buescher ◽  
JI Gallin
Keyword(s):  
Cell Size ◽  
Graft Versus Host Disease ◽  
Radiation Effects ◽  
Human Monocyte ◽  
Cell Protein ◽  
Human Monocytes ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ldh Release

Prior to administration, leukocyte transfusions are commonly irradiated with up to 5,000 R to eliminate lymphocytes and thereby prevent graft- versus-host disease in the recipient. It has been widely believed that phagocytes are resistant to this irradiation. In a recent report, we noted that phagocyte oxidative metabolism was compromised during preparation of white cells for transfusion. As part of our effort to examine the basis for this inhibition of phagocyte function during white cell preparation, we assessed the effects of irradiation on the long-lived monocytes that have been shown to persist at inflammatory foci posttransfusion. Human monocytes were irradiated for up to 3 min, receiving 2,500–5,000 R. This irradiation damaged human monocytes, significantly decreasing their in vitro survival for the first 3 wk of culture (p less than 0.02, irradiated versus control survival), and growth as assessed by two-dimensional cell size measurements during the first 2 wk of culture (p less than 0.01, irradiated versus control growth). Despite smaller cell size, total cell protein was significantly increased over time in irradiated cultures (p less than 0.001, irradiated versus control total protein per cell). Extracellular release of lysozyme and beta-glucuronidase per cell was not affected by irradiation, but extracellular lactate dehydrogenase (LDH) release was significantly increased after irradiation (p less than 0.001, irradiated versus control LDH release). Irradiated monocytes killed Listeria monocytogenes at a slower rate than the nonirradiated controls (p less than 0.05, irradiated versus control rate of killing). Thus, the data indicate that irradiation in doses used to prevent graft- versus-host disease in leukocyte transfusion recipients has a deleterious effect on in vitro human monocyte survival and function.

scholarly journals Monocytes and macrophages synthesize and secrete thrombospondin

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 79-84 ◽  
Author(s):  
EA Jaffe ◽  
JT Ruggiero ◽  
DJ Falcone
Keyword(s):  
Culture Medium ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Human Monocyte ◽  
Peritoneal Macrophages ◽  
Time Dependent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Monocytes And Macrophages

hrombospondin, one of the major glycoproteins released from alpha- granules of thrombin-stimulated platelets, is a disulfide-linked trimer of 160,000-dalton subunits. Cultured human monocytes secreted thrombospondin (determined by an enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (1.45 micrograms/10(6) cells/24 hr); secretion was totally blocked by cycloheximide (1 microgram/mL). 35S-thrombospondin was isolated from 35S-methionine-labeled human monocyte postculture medium with rabbit polyclonal anti-thrombospondin coupled to protein A-Sepharose. The immunoisolated 35S-thrombospondin migrated in sodium dodecyl sulfate- polyacrylamide gels after reduction with a molecular weight of 159,000. Similar results were obtained using mouse resident peritoneal macrophages. Elicited peritoneal macrophages harvested from mice pretreated with endotoxin, casein, or thioglycollate secreted much less thrombospondin than did resident macrophages harvested from control mice. Thus, monocytes and macrophages from two different species synthesize and secrete thrombospondin, and the rate of synthesis of thrombospondin appears to depend on the state of activation of the cells.

scholarly journals Monocytes and macrophages synthesize and secrete thrombospondin

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 79-84 ◽  
Author(s):  
EA Jaffe ◽  
JT Ruggiero ◽  
DJ Falcone
Keyword(s):  
Culture Medium ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Human Monocyte ◽  
Peritoneal Macrophages ◽  
Time Dependent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Monocytes And Macrophages

Abstract hrombospondin, one of the major glycoproteins released from alpha- granules of thrombin-stimulated platelets, is a disulfide-linked trimer of 160,000-dalton subunits. Cultured human monocytes secreted thrombospondin (determined by an enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (1.45 micrograms/10(6) cells/24 hr); secretion was totally blocked by cycloheximide (1 microgram/mL). 35S-thrombospondin was isolated from 35S-methionine-labeled human monocyte postculture medium with rabbit polyclonal anti-thrombospondin coupled to protein A-Sepharose. The immunoisolated 35S-thrombospondin migrated in sodium dodecyl sulfate- polyacrylamide gels after reduction with a molecular weight of 159,000. Similar results were obtained using mouse resident peritoneal macrophages. Elicited peritoneal macrophages harvested from mice pretreated with endotoxin, casein, or thioglycollate secreted much less thrombospondin than did resident macrophages harvested from control mice. Thus, monocytes and macrophages from two different species synthesize and secrete thrombospondin, and the rate of synthesis of thrombospondin appears to depend on the state of activation of the cells.

scholarly journals Radiation effects on cultured human monocytes and on monocyte-derived macrophages

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1402-1407 ◽  
Author(s):  
ES Buescher ◽  
JI Gallin
Keyword(s):  
Cell Size ◽  
Graft Versus Host Disease ◽  
Radiation Effects ◽  
Human Monocyte ◽  
Cell Protein ◽  
Human Monocytes ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ldh Release

Abstract Prior to administration, leukocyte transfusions are commonly irradiated with up to 5,000 R to eliminate lymphocytes and thereby prevent graft- versus-host disease in the recipient. It has been widely believed that phagocytes are resistant to this irradiation. In a recent report, we noted that phagocyte oxidative metabolism was compromised during preparation of white cells for transfusion. As part of our effort to examine the basis for this inhibition of phagocyte function during white cell preparation, we assessed the effects of irradiation on the long-lived monocytes that have been shown to persist at inflammatory foci posttransfusion. Human monocytes were irradiated for up to 3 min, receiving 2,500–5,000 R. This irradiation damaged human monocytes, significantly decreasing their in vitro survival for the first 3 wk of culture (p less than 0.02, irradiated versus control survival), and growth as assessed by two-dimensional cell size measurements during the first 2 wk of culture (p less than 0.01, irradiated versus control growth). Despite smaller cell size, total cell protein was significantly increased over time in irradiated cultures (p less than 0.001, irradiated versus control total protein per cell). Extracellular release of lysozyme and beta-glucuronidase per cell was not affected by irradiation, but extracellular lactate dehydrogenase (LDH) release was significantly increased after irradiation (p less than 0.001, irradiated versus control LDH release). Irradiated monocytes killed Listeria monocytogenes at a slower rate than the nonirradiated controls (p less than 0.05, irradiated versus control rate of killing). Thus, the data indicate that irradiation in doses used to prevent graft- versus-host disease in leukocyte transfusion recipients has a deleterious effect on in vitro human monocyte survival and function.

Ultrastructural observations of human monocytes infected with HIV-1

1991 ◽  
Vol 49 ◽  
pp. 108-109
Author(s):  
James K. Koehler ◽  
Steven G. Reed ◽  
Joao S. Silva
Keyword(s):  
Gradient Centrifugation ◽  
Virus Production ◽  
Human Monocyte ◽  
Red Cross ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Hiv Replication ◽  
Electron Microscopic ◽  
Ultrastructural Studies ◽  
Hiv 1

As part of a larger study involving the co-infection of human monocyte cultures with HIV and protozoan parasites, electron microscopic observations were made on the course of HIV replication and infection in these cells. Although several ultrastructural studies of the cytopathology associated with HIV infection have appeared, few studies have shown the details of virus production in “normal,” human monocytes/macrophages, one of the natural targets of the virus, and suspected of being a locus of quiescent virus during its long latent period. In this report, we detail some of the interactions of developing virons with the membranes and organelles of the monocyte host.Peripheral blood monocytes were prepared from buffy coats (Portland Red Cross) by Percoll gradient centrifugation, followed by adherence to cover slips. 90-95% pure monocytes were cultured in RPMI with 5% non-activated human AB serum for four days and infected with 100 TCID50/ml of HIV-1 for four hours, washed and incubated in fresh medium for 14 days.

scholarly journals Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification.

1991 ◽  
Vol 11 (11) ◽  
pp. 5389-5397 ◽  
Author(s):  
P Yaciuk ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Cell Function ◽  
Rat Kidney ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Cell Protein ◽  
Cycle Phase ◽  
Resting Cells ◽  
Kidney Cells ◽  
Nuclear Phosphoprotein

Binding of a 300-kDa host cell protein (p300) is tightly correlated with the ability of the adenovirus E1A products to induce quiescent baby rat kidney cells to proliferate. We have generated rabbit polyclonal antibodies against p300 to characterize this protein further. We have found p300 to be a nuclear phosphoprotein that is actively synthesized in both quiescent and proliferating baby rat kidney cells. In partially purified mitotic cell populations, we observe a form of p300 with decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that shares a nearly identical partial proteolytic digest pattern with p300. The slower-migrating form of p300 is greatly reduced by treating immune complexes with potato acid phosphatase. The relative stability and presence of p300 even in resting cells suggests that p300 has a basal cell function, but the appearance of differentially modified forms during the cell cycle suggests the possibility that p300 function is modulated specifically in growing cells.

Separation of Heavy Metal Ions from the Solution Obtained by Leaching Low-Grade Pyrolusite with Pyrite and H2SO4

2013 ◽  
Vol 773 ◽  
pp. 283-288
Author(s):  
Xing Zou ◽  
Xiang Quan Chen ◽  
Hai Chao Xie ◽  
Xiao Dan Qiu
Keyword(s):  
Heavy Metal ◽  
Metal Ions ◽  
Heavy Metal Ions ◽  
Separation Efficiency ◽  
Ph Value ◽  
Sulfate Solution ◽  
Ion Concentration ◽  
Manganese Sulfate ◽  
Low Grade ◽  
High Concentration

The manganese sulfate solution leached from low-grade pyrolusite with pyrite and H2SO4 contains heavy metal ions of high concentration, influencing the quality of the final products of manganese compounds and causing manganese ions not to be electrolyzed. The present study was focused on the separation of Co, Ni and Zn ions from the leached solution with BaS. By controlling the pH value at 5.0-6.5, temperature at 50-60°C, reaction time at 15 min and mixing velocity at 78 rpm, the heavy metal ions could be separated effectively. Under the above optimized conditions, the ion concentration of Co, Ni, and Zn in the solution was reduced to 0.06 mg.L-1, 0.27mg.L-1 and 0.01mg.L-1, and the separation efficiency was 99.72%, 99.18% and 99.9% respectively. The obtained pure solution meets the demands of manganese electrowinning.

scholarly journals An epigenetic GPI anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julia Wegner ◽  
Thomas Zillinger ◽  
Thais Schlee-Guimaraes ◽  
Eva Bartok ◽  
Martin Schlee
Keyword(s):  
Single Cell ◽  
Human Monocyte ◽  
Surface Expression ◽  
Human Monocytes ◽  
Innate Immune Responses ◽  
Loss Of Function ◽  
Cell Cloning ◽  
Gpi Anchor ◽  
Single Cell Cloning ◽  
Antigen Presenting

AbstractAntigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.

Abscisic acid and low temperature induced polypeptide changes in alfalfa (Medicago sativa) cell suspension cultures

1986 ◽  
Vol 64 (11) ◽  
pp. 2758-2763 ◽  
Author(s):  
Albert J. Robertson ◽  
Lawrence V. Gusta
Keyword(s):  
Abscisic Acid ◽  
Low Temperature ◽  
Medicago Sativa ◽  
Cell Suspension ◽  
Sodium Dodecyl ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Total Cell ◽  
Cell Protein ◽  
Major Protein

Changes in extracellular, cellular, and subcellular proteins during abscisic acid and low temperature induced cold hardening of alfalfa (Medicago sativa L. cv. Wisconsin 22C) cell suspension cultures were investigated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Extracellular proteins from 4- to 6-day-old ABA and low temperature grown alfalfa cells showed decreased electrophoretic mobilities, lacked a 190-kDa glycoprotein, and had reduced amounts of four other polypeptides. In total cell protein analyses, a 42-kDa protein was enriched in both ABA and low temperature treated alfalfa cells. Several proteins increased or induced by exogenous ABA treatment were identified in the extracellular (12.5 and 13 to 15 kDa), total cell and cell wall (24 kDa), and soluble (20, 37, and 41 kDa) fractions. However, no major protein changes were resolved by one-dimensional electrophoretic analyses of crude membrane proteins.

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