Specific Binding of Phosphorylated Non-Histone Chromatin Proteins to Deoxyribonucleic Acid

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.

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Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162661 ◽

1996 ◽

Vol 54 ◽

pp. 40-41

Author(s):

László G. Kömüves

Keyword(s):

San Francisco ◽

Colloidal Gold ◽

Specific Binding ◽

Ultrastructural Localization ◽

Ultrastructural Level ◽

Uranyl Acetate ◽

Adhesive Tape ◽

Distilled Water ◽

Capillary Action ◽

Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

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On the Role of Transferrin in 67Ga Uptake by Tumor Cells

Nuklearmedizin ◽

10.1055/s-0038-1629447 ◽

1988 ◽

Vol 27 (04) ◽

pp. 151-153

Author(s):

P. Thouvenot ◽

F. Brunotte ◽

J. Robert ◽

L. J. Anghileri

Keyword(s):

Specific Binding ◽

Subcellular Fractionation ◽

Animal Blood ◽

Tumor Bearing ◽

Cell Structures ◽

The Difference ◽

Sarcoma Cells ◽

In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

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Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647851 ◽

1975 ◽

Vol 33 (03) ◽

pp. 573-585 ◽

Cited By ~ 20

Author(s):

Masahiro Iwamoto

Keyword(s):

Tranexamic Acid ◽

Affinity Chromatography ◽

Dissociation Constant ◽

Factor Xiii ◽

Specific Binding ◽

The Other ◽

Inhibitory Mechanism ◽

Binding Studies ◽

Similar Affinity ◽

Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

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Concanavalin A-induced Aggregation of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647887 ◽

1975 ◽

Vol 33 (02) ◽

pp. 354-360 ◽

Cited By ~ 4

Author(s):

Heinrich Patscheke ◽

Reinhard Brossmer

Keyword(s):

Concanavalin A ◽

Binding Capacity ◽

Specific Binding ◽

Blood Platelets ◽

Residual Effect ◽

Independent Effect ◽

Con A ◽

Experimental Conditions ◽

Main Effect ◽

Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

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Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648973 ◽

1994 ◽

Vol 72 (06) ◽

pp. 848-855 ◽

Cited By ~ 34

Author(s):

Dzung The Le ◽

Samuel I Rapaport ◽

L Vijaya Mohan Rao

Keyword(s):

Cell Surface ◽

Tissue Factor ◽

Factor Viia ◽

Cell Damage ◽

Specific Binding ◽

Surface Membrane ◽

Annexin V ◽

Factor X ◽

Binding Studies ◽

Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

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Autoantibodies to Thrombomodulin: Development of an Enzyme Immunoassay and a Survey of their Frequency in Patients with the Lupus Anticoagulant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648482 ◽

1992 ◽

Vol 67 (05) ◽

pp. 507-509 ◽

Cited By ~ 16

Author(s):

John Gibson ◽

Margaret Nelson ◽

Ross Brown ◽

Hatem Salem ◽

Harry Kronenberg

Keyword(s):

Enzyme Immunoassay ◽

Lupus Anticoagulant ◽

Specific Binding ◽

Technical Problem ◽

Serum Samples ◽

Citrate Buffer ◽

The Mean ◽

Sample Dilution ◽

Negative Population ◽

Serum Components

SummaryIn order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (± SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 ± 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.

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