Sedimentation and Actinomycin D Binding Studies of Partially Denatured Crab dAT*

Biochemistry ◽  
1966 ◽  
Vol 5 (5) ◽  
pp. 1753-1759 ◽  
Author(s):  
Jack M. Widholm ◽  
James Bonner
Keyword(s):  
Actinomycin D ◽  
Binding Studies

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Lectin Binding Studies on RNA Tumor Virus-Infected Cells

1975 ◽  
Vol 33 ◽  
pp. 326-327
Author(s):  
D. C. Hixson
Keyword(s):  
Binding Sites ◽  
Lectin Binding ◽  
Culture Conditions ◽  
3T3 Cells ◽  
Binding Studies ◽  
Con A ◽  
Microscopic Studies ◽  
Tumor Virus ◽  
Infected Cells ◽  
Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

Towards understanding the mechanism of action of mithramycin and its analogues: dimerization and DNA binding studies

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
S Weidenbach ◽  
C Hou ◽  
JM Chen ◽  
OV Tsodikov ◽  
J Rohr
Keyword(s):  
Dna Binding ◽  
Mechanism Of Action ◽  
Binding Studies ◽  
Dna Binding Studies

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

1975 ◽  
Vol 33 (03) ◽  
pp. 573-585 ◽  
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.

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

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
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.

The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

1997 ◽  
Vol 77 (05) ◽  
pp. 0949-0954 ◽  
Author(s):  
J Prins ◽  
F R Lues ◽  
Y Y van der Hoek ◽  
J J.P Kastelein ◽  
B N Bouma ◽  
...  
Keyword(s):  
Case Control Study ◽  
Amino Acid Position ◽  
Case Control ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Apolipoprotein A ◽  
Significant Independent Risk Factor ◽  
And Gender ◽  
Control Study

SummaryElevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr12-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (DesafibTM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafibTM-X was observed for the Pro12-subtype Lp(a).

ANALYSIS OF THE BIPHASIC ACTION OF GROWTH HORMONE IN VITRO ON THE RAT DIAPHRAGM

1968 ◽  
Vol 57 (3_Suppl) ◽  
pp. S19-S35 ◽  
Author(s):  
Å. Hjalmarson
Keyword(s):  
Growth Hormone ◽  
Actinomycin D ◽  
Accumulation Rate ◽  
Inhibitory Effect ◽  
Bovine Growth Hormone ◽  
Rat Diaphragm ◽  
Dual Nature ◽  
Hypophysectomized Rats ◽  
D 20

ABSTRACT In vitro addition of bovine growth hormone (GH) to intact hemidiaphragms from hypophysectomized rats has previously been found to produce both an early stimulatory effect lasting for 2—3 hours and a subsequent late inhibitory effect during which the muscle is insensitive to further addition of GH (Hjalmarson 1968). These effects on the accumulation rate of α-aminoisobutyric acid (AIB) and D-xylose have been further studied. In presence of actinomycin D (20 μg/ml) or puromycin (100 μg/ml) the duration of the stimulatory effect of GH (25 μg/ml) was prolonged to last for at least 4—5 hours and the late inhibitory effect was prevented. Similar results were obtained when glucose-free incubation medium was used. Preincubation of the diaphragm at different glucose concentrations (0—5 mg/ml) for 3 hours did not change the GH sensitivity. Addition of insulin at start of incubation could not prevent GH from inducing its late inhibitory effect, while dexamethasone seemed to potentiate this effect of GH. Furthermore, adrenaline was found to decrease the uptake of AIB-14C and D-xylose-14C in the diaphragm, but not to change the sensitivity of the muscle to GH. Preincubation of the diaphragm for 3 hours with puromycin in a concentration of 200 μg/ml markedly decreased the subsequent basal uptake of both AIB-14C and D-xylose-14C, in the presence of puromycin, and abolished the stimulatory effect of GH on the accumulation of AIB-14C. However, the effect of GH on the accumulation of D-xylose-14C was unchanged. The present observations are discussed and evaluated in relation to various mechanisms of GH action proposed to explain the dual nature of the hormone.

CONTRIBUTION TO THE EXISTENCE OF REGULATORY MECHANISMS AT THE ADRENAL LEVEL

1972 ◽  
Vol 70 (4) ◽  
pp. 741-757
Author(s):  
Otto Linèt
Keyword(s):  
Orotic Acid ◽  
Adrenal Glands ◽  
Actinomycin D ◽  
Delay Period ◽  
Regulatory Mechanisms ◽  
Leucine Incorporation ◽  
Total Period ◽  
Free Media

ABSTRACT Rat adrenal glands atrophied by the administration of cortisol acetate in vivo were used as a model for the study of early metabolic processes occurring in vitro. Atrophied adrenals incubated in the presence of 14C-leucine incorporated subnormal quantities of this amino acid per mg of protein for the first 120 min. When the incubation lasted for a total period of 180 or 240 min a supranormal rise in the 14C-leucine incorporation was observed. Similar changes occurred with some delay with regard to corticosterone production as expressed per 100 mg of tissue. No differences in 14C-leucine incorporation were observed between the control and atrophied adrenals in vivo. Homogenates from atrophied glands incorporated 14C-leucine to a greater extent than the control homogenates. The in vitro incorporation of 14C-orotic acid into the RNA was also higher in atrophied adrenals. The in vitro use of actinomycin D, cycloheximide and amphenone indicated that corticosterone production depended on the incorporation of 14C-leucine. The addition of cortisol to the incubation media markedly decreased the enhancement of 14C-lysine incorporation into the protein of atrophied adrenals. These, as well as additional results suggest rebound phenomena: once atrophic adrenals are transferred to cortisol-free media, reparative processes begin after a delay period. Such phenomena seem to be mediated by regulatory mechanisms at the adrenal level.

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