Coexistence of two biochemically distinct phospholipase A2 activities in human platelet, monocyte, and neutrophil

1993 ◽  
Vol 71 (7-8) ◽  
pp. 331-339 ◽  
Author(s):  
Lisa A. Marshall ◽  
Amy Roshak
Keyword(s):  
Arachidonic Acid ◽  
Specific Activity ◽  
Human Platelet ◽  
High Specific Activity ◽  
Cell Types ◽  
Phospholipase A ◽  
Joint Fluid ◽  
Synovial Joint ◽  
Monocytic Cell ◽  
Activity Measurements

The cell-associated phospholipase A2 (PLA2) activities of the human platelet, neutrophil, and monocyte were simultaneously characterized, utilizing the biochemical differences observed between the 14 kDa (kilodalton), type II PLA2 isolated from inflammatory human synovial joint fluid (HSF) and the arachidonic acid (AA) specific, 85-kDa high molecular mass (HMM) PLA2 isolated from the cytosol of a U937 monocytic cell line. The HSF PLA2 can be distinguished from the HMM PLA2 by its resistance to acid treatment, sensitivity to a sulfhydryl reducing agent, lack of preference for the fatty acid on the sn-2 position of phospholipid substrate, and inhibition by the C-7 phosphonate–phospholipid transition-state PLA2 inhibitor. Evaluation of all three cell types revealed that HMM-like PLA2 activity was found predominantly in the cytosolic fractions, although detection in neutrophil cytosol required more concentrated preparations and the use of high specific activity [3H]AA-labeled Escherichia coli. HSF-PLA2-like activity was measured in microsomal and cytosolic fractions of all three cell types, but was found in neutrophil cytosol only after treatment with acid. Further HMM-PLA2-specific interfering agents in neutrophil cytosol were observed and exemplifies one problem in assigning the existence of this enzyme in crude broken cell preparations using activity measurements alone. The role that these two enzymes play in eicosanoid production of the respective cell types remains to be studied.Key words: phospholipase A2, arachidonic acid, neutrophil, platelet, monocyte, cytosol, microsome.

scholarly journals Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 1002-1005 ◽  
Author(s):  
J Cashman ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Growth Medium ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Specific Cell ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Marrow Cultures

We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

scholarly journals Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine

1979 ◽  
Vol 184 (2) ◽  
pp. 283-290 ◽  
Author(s):  
V A Ziboh ◽  
J T Lord
Keyword(s):  
Arachidonic Acid ◽  
Prostaglandin E2 ◽  
Specific Activity ◽  
Feedback Regulation ◽  
Phospholipase A ◽  
Particulate Fraction ◽  
Prostaglandin F2 Alpha ◽  
Low Concentrations ◽  
Freeze Dried ◽  
Hydrolysis Of

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.

scholarly journals A Secondary Standard for Radiocarbon Dating

Radiocarbon ◽  
1983 ◽  
Vol 25 (2) ◽  
pp. 541-546 ◽  
Author(s):  
Fernando E Angiolini ◽  
Miguel C Albero
Keyword(s):  
Oxalic Acid ◽  
Radiocarbon Dating ◽  
Specific Activity ◽  
Barium Carbonate ◽  
Statistical Tests ◽  
High Specific Activity ◽  
Secondary Standard ◽  
Isotopic Dilution ◽  
Acid Attack ◽  
Activity Measurements

The preparation and calibration of a secondary standard for the INGEIS Radiocarbon Dating Laboratory are presented. This standard is barium carbonate with a specific activity almost twice that of NBS oxalic acid. It was prepared from BaCO3 with high specific activity and commercial potassium carbonate by an isotopic dilution technique. The advantages of this standard are: 1) the preparation is simple and can be achieved with ordinary labware; 2) the production of CO2 by acid attack from this carbonate shows minimum isotopic fractionation. At least, it has less fractionation than wet oxidation of oxalic acid, the problems of which are described in the literature. This standard ensures better reproducibility in activity measurements; 3) despite some problems of activity exchange with atmospheric CO2 concerning carbonates, measurements of activity over a period of about two years have shown no significant deviation from the mean value. A tentative explanation of this phenomenon is also given. The activity ratio between BaCO3 and NBS oxalic acid is given with its error, and the statistical tests used in the calibration are briefly explained. Finally, a control chart for the activity of the standard over a long period is drawn, showing non-significant deviation and supporting the usefulness of this standard.

scholarly journals Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 1002-1005 ◽  
Author(s):  
J Cashman ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Growth Medium ◽  
Specific Activity ◽  
High Specific Activity ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Specific Cell ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Marrow Cultures

Abstract We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

scholarly journals ISOLATION OF PARIETAL CELLS FROM GLUTARALDEHYDE-FIXED RABBIT STOMACH

1972 ◽  
Vol 20 (8) ◽  
pp. 634-643 ◽  
Author(s):  
SIN HANG LEE
Keyword(s):  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Cell Types ◽  
Celiac Artery ◽  
Parietal Cells ◽  
Adult Rabbit ◽  
Isolated Cells ◽  
Discontinuous Sucrose Gradient ◽  
Final Yield

A method is introduced to isolate and purify parietal cells from the rabbit stomach which has been briefly perfused with 1% glutaraldehyde via the celiac artery. The fixed mucosa is initially digested with 1% trypsin and subsequently with 1% collagenase solution. The parietal cells are more resistant to the effects of these proteolytic enzymes than the other cell types in the gastric mucosa and can be recovered and purified by centrifugation in a discontinuous sucrose gradient. Glutaraldehyde fixation reduces the tendency of cell clumping and, therefore, facilitates isolation of the separated cells. The final yield from each adult rabbit stomach varies from 0.1 to 0.4 ml packed parietal cells of about 97% purity determined by nuclear counts. The nuclei and mitochondria of most of the isolated cells appear to be intact on electron micrographs, but the cell membrane is focally disrupted; the nuclear deoxyribonucleic acid (DNA) can be stained with Feulgen technique and extracted with perchloric acid for quantitative determination. Biochemical studies on incorporation of thymidine-3H into DNA of the parietal cells have been carried out in normal young adult rabbits and in rabbits under chronic histamine stimulation. The results show that there is no significant incorporation of thymidine-3H by mature parietal cells in any of the two groups of animals, although the whole mucosa DNA hydrolysates always exhibit a high specific activity.

A New Method for Plasminogen Standardization

1968 ◽  
Vol 20 (03/04) ◽  
pp. 548-554
Author(s):  
J Gajewski ◽  
G Markus
Keyword(s):  
Proteolytic Activity ◽  
Specific Activity ◽  
Molar Ratio ◽  
Sharp Transition ◽  
Equivalence Point ◽  
Constant Amount ◽  
Residual Level ◽  
Activity Measurements ◽  
Complete Saturation ◽  
Human Plasminogen

SummaryA method for the standardization of human plasminogen is proposed, based on the stoichiometric interaction between plasminogen and streptokinase, resulting in inhibition of proteolytic activity. Activation of a constant amount of plasminogen with increasing amounts of streptokinase yields linearly decreasing activities, as a function of streptokinase, with a sharp transition to a constant residual level. The point of transition corresponds to complete saturation of plasmin with streptokinase in a 1:1 molar ratio, and is therefore a measure of the amount of plasminogen present initially, in terms of streptokinase equivalents. The equivalence point is independent of the kind of protein substrate used, buffer, pH, length of digestion and, within limits, temperature. The method, therefore, is not subject to the variations commonly encountered in the usual determination based on specific activity measurements.

Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

1981 ◽  
Vol 46 (02) ◽  
pp. 538-542 ◽  
Author(s):  
R Pilo ◽  
D Aharony ◽  
A Raz
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Unsaturated Fatty Acids ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers
Keyword(s):  
Snake Venom ◽  
Thrombolytic Agent ◽  
Sodium Citrate ◽  
Specific Activity ◽  
High Specific Activity ◽  
Citrate Solution ◽  
Clotting Factors ◽  
Activation Products ◽  
Russell’S Viper ◽  
Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

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