scholarly journals The binding of adenosine(5′)tetraphospho(5′)adenosine to calf thymus histones measured by non-equilibrium dialysis

1987 ◽  
Vol 246 (3) ◽  
pp. 681-686 ◽  
Author(s):  
G Just ◽  
E Holler
Keyword(s):  
Ionic Strength ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Physiological Saline ◽  
Histone H1 ◽  
The Other ◽  
Competition Assay ◽  
Calf Thymus ◽  
Non Equilibrium

Binding of adenosine(5′)tetraphospho(5′)adenosine (Ap4A) to histones of calf thymus was investigated by non-equilibrium dialysis. Histone H1 interacts with the dinucleotide via two strong sites and competes with Mg2+ ions. Intrinsic dissociation constants were 1.6 +/- 0.1 microM and 11 +/- 1 microM for zero and 0.4 mm-Mg2+ concentration respectively. Binding of poly(dT) and of other nucleotides to histone H1 was measured in an [3H]Ap4A-competition assay. The tendency to form complexes among nucleotides was highest for bisnucleoside tetraphosphates and decreased in the order poly(dT) greater than or equal to Ap4A approximately Gp4G greater than Ap4 much greater than Ap3A approximately Ap5A greater than or equal to ATP, GTP and dTTP. The co-ordination complex derived from Ap4A and cis-diammine-dichloroplatinum(II) was not reactive. The other histones of calf thymus also bound Ap4A with affinities decreasing in the order H4 approximately H3 greater than H1 greater than H2b greater than H2a. Ap4A stimulated the exchange of histone H1 between nucleosomes, but this effect was referred to ionic strength. It did not bind to assembled nucleosomes. Binding of Ap4A to histone H1 was decreased by salt (NaCl). At physiological saline concentration the value of the dissociation constant is commensurable with the value of the Ap4A concentration in the nucleus and thus indicative of complex-formation in vivo.

The selective extraction of histones from rye chromatin

1976 ◽  
Vol 54 (9) ◽  
pp. 765-771 ◽  
Author(s):  
Hélène LaRue ◽  
Dominick Pallotta
Keyword(s):  
Selective Extraction ◽  
Histone H1 ◽  
The Other ◽  
Calf Thymus

The selective extraction of histones from rye chromatin was studied using three different methods. Extractions with NaCl–phosphate buffers atpH 5.5 gave results similar to those already obtained with other types of chromatin. Histone H1 was selectively extracted with 0.6 M NaCl –0.001 M PO4, while the selectivity of dissociation of the other fractions was reduced at higher NaCl concentrations. The use of phosphate–urea buffers at pH 5.5 also revealed that the histones were dissociated at the same concentrations as were calf thymus histones. Histone H1 was extracted with 0.5 M PO4 – 1 M urea; H1, H2A, and H2B were extracted with 0.8 M PO4 – 2 M urea; and all histones were removed with 0.8 M PO4 –5.3 M urea. It was, however, observed that the dissociated rye histones H2A and H2B were unstable in these buffers. This instability was maximum in the presence of 3 M urea, where both histones were absent from the extracted proteins and the residual nucleoproteins. Finally, a solution of 30% ethanol −0.35 M NaCl – 6 M urea produced a rye nucleoprotein fraction containing only histone H1.

Monoclonal Antibodies to Human Fibrin: Interaction with other Animal Fibrins

1996 ◽  
Vol 75 (04) ◽  
pp. 595-599 ◽  
Author(s):  
Tracey Edgell ◽  
F McEvoy ◽  
P Webbon ◽  
P J Gaffney
Keyword(s):  
Monoclonal Antibodies ◽  
Dissociation Constant ◽  
Dissociation Constants ◽  
The Other ◽  
Limited Data ◽  
Thrombolytic Agents ◽  
Thrombus Imaging ◽  
Fibrin Clots

SummaryFour monoclonal antibodies have previously been raised in our laboratory for possible use in thrombus imaging and the targeting of thrombolytic agents. These antibodies were raised to various human fibrin-related immunogens and each antibody had been selected for its specificity towards fibrin and not fibrinogen. To study further these antibodies in animal circulation models both in vivo and in vitro, their selectivity towards human fibrin as opposed to other animal fibrins was examined. In this study dissociation constants for each antibody with each of six species fibrins (human, baboon, pig, dog, sheep, and rabbit) were estimated using both fibrin clots and monolayers. Some limited data were also obtained with Sepharose-fibrin. Of the antibodies two (denoted 12B3B10 and 12B3A11) are seen to bind almost exclusively to human fibrin with dissociation constants of about 8 × 10−10 M using fibrin clots and monolayers. These same two mabs bound to baboon fibrin with a dissociation constant of 2 × 10−9 M, while neither displayed significant levels of binding to the fibrins from dog, pig, sheep and rabbit. The other two antibodies investigated (1H10 and 5F3) were found to bind well to fibrins of human, baboon, pig and dog, with dissociation constants in the range of 1.4-4.2 × 10−9 M. However neither 1H10 nor 5F3 displayed significant recognition of sheep and rabbit fibrins. Both 1H10 and 5F3 were also found by means of competitive ELISA’s to retain their selectivity to baboon, dog and pig fibrins in the presence of their respective fibrinogens.

Exogenous histone H1 injection into mitotic cells disrupts synchronous progression of mitotic events by delaying chromosome decondensation

1994 ◽  
Vol 107 (3) ◽  
pp. 693-701 ◽  
Author(s):  
Y. Matsuoka ◽  
S. Takechi ◽  
T. Nakayama ◽  
Y. Yoneda
Keyword(s):  
Mitotic Spindle ◽  
Protein Transport ◽  
Type A ◽  
Histone H1 ◽  
Type B ◽  
Lamin B ◽  
Calf Thymus ◽  
Exogenous Histone ◽  
Chromosome Decondensation

At the end of open mitosis, chromosome decondensation, nuclear envelope re-formation and reassembly of interphase microtubules following mitotic spindle dissociation occur coordinately. To determine whether these events progress only synchronously in vivo, we delayed chromosome decondensation by injecting of exogenous proteins into the mitotic rat kangaroo kidney epithelium (PtK2) cells. When histone H1 purified from calf thymus was injected at prometaphase, chromosome condensation was prolonged for several hours, and sister chromatid separation and cytokinesis did not occur. However, interphase microtubules reassembled and lamin B-positive structures re-formed around the condensed chromosomes. Exactly the same results were obtained on injection of bacterially expressed H1. Kinetic experiments showed that there were two types of lamin B-positive structures. One type (type A) was stained uniformly with anti-lamin B antibodies. The other (type B) showed peripheral lamin B staining; that is, the normal interphase staining pattern, and was found to be competent for nuclear protein transport. As the chromosomes decondensed, the amount of type A decreased and that of type B increased. However, even cells containing highly condensed chromosomes had both type A and type B. From these results, we conclude that the re-formation of microtubules and reassembly of a nuclear transport-competent envelope do not depend on chromosome decondensation.

Nucleosomes are assembled into discrete size structures by histone H1 in vitro

1986 ◽  
Vol 64 (5) ◽  
pp. 463-473 ◽  
Author(s):  
Teni Boulikas
Keyword(s):  
Ionic Strength ◽  
Histone H1 ◽  
Higher Order ◽  
Third Order ◽  
Calf Thymus ◽  
Exogenous Histone ◽  
Structural Subunit ◽  
Chromatin Structural ◽  
Low Ionic Strength

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller super-nucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supemucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.

scholarly journals Action of Dinitrophenyl Amino Acids on Skeletal Muscle Proteins II. Absorption of Bis-Dinitrophenyl-Lysine By Light and Heavy Meromyosins and by Light Meromyosin Fraction I

1968 ◽  
Vol 21 (1) ◽  
pp. 141
Author(s):  
RW Burley ◽  
WJH Jackson ◽  
Jean P Robertson
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Ionic Strength ◽  
High Speed ◽  
Equilibrium Dialysis ◽  
The Other ◽  
Muscle Proteins ◽  
Heavy Meromyosin ◽  
Skeletal Muscle Proteins ◽  
Fraction I

The quantity of bis(2,4-dinitrophenyl)-L-Iysine (abbreviation bis-DNP-Iysine) absorbed at 5�C by myosin and meromyosins of rabbit skeletal muscle was estimated in phosphate buffer (pH 7, ionic strength 0�5) by two methods, one based on equilibrium dialysis, the other on high-speed centrifugation. According to both methods, heavy meromyosin absorbed more of the reagent than did the same weight of the parent myosin; light meromyosin absorbed less, and light meromyosin fraction I absorbed less still. There were, however, relatively large quantitative differences between the two methods, possibly because of an effect of the slightly different conditions of treatment.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Policy analysis and complexity – a non-equilibrium approach for integrated water management

1995 ◽  
Vol 31 (8) ◽  
pp. 301-309 ◽  
Author(s):  
Govert D. Geldof
Keyword(s):  
Water Management ◽  
Control System ◽  
Planning Process ◽  
The Other ◽  
Integrated Water Management ◽  
Equilibrium Approach ◽  
History Of ◽  
The One ◽  
Groundwater Policy ◽  
Non Equilibrium

In integrated water management, the issues are often complex by nature, they are capable of subjective interpretation, are difficult to express in standards and exhibit many uncertainties. For such issues, an equilibrium approach is not appropriate. A non-equilibrium approach has to be applied. This implies that the processes to which the integrated issue pertains, are regarded as “alive”’. Instead of applying a control system as the model for tackling the issue, a network is used as the model. In this network, several “agents”’ are involved in the modification, revision and rearrangement of structures. It is therefore an on-going renewal process (perpetual novelty). In the planning process for the development of a groundwater policy for the municipality of Amsterdam, a non-equilibrium approach was adopted. In order to do justice to the integrated character of groundwater management, an approach was taken, containing the following features: (1) working from global to detailed, (2) taking account of the history of the system, (3) giving attention to communication, (4) building flexibility into the establishing of standards, and (5) combining reason and emotions. A middle course was sought, between static, rigid but reliable on the one hand; dynamic, flexible but vague on the other hand.

Endo-D-galacturonanase immobilized by adsorption on porous polyethyleneterephthalate

1982 ◽  
Vol 47 (10) ◽  
pp. 2716-2723 ◽  
Author(s):  
Lubomíra Rexová-Benková ◽  
Jiřina Omelková ◽  
Vladimír Kubánek
Keyword(s):  
Ionic Strength ◽  
High Stability ◽  
Relative Activity ◽  
Operational Stability ◽  
The Other ◽  
Fixation Strength ◽  
Action Pattern ◽  
Salt Solutions ◽  
Buffer Solution ◽  
Degradation Degree

Endo-D-galacturonanase of Aspergillus sp. was irreversibly adsorbed on polyethyleneterephthalate in an acetate 0.1 mol l-1 buffer solution of pH 4.2. Immobilization of the enzyme resulted in lowering of its activity, the measure of which depended on the amount of the enzyme fixed on the carrier. The highest relative activity (42.4%) had the preparation containing 5.25 mg of the enzyme per 1 g of the carrier. The velocity and intensity of the sorption of the enzyme depended on the ionic strength of the medium, whilst pH, on the other hand, was of no influence. Endo-D-galacturonanase immobilized in a 0.1 mol l-1 buffer was characteristic a) of its fixation strength in salt solutions of various ionic strength and pH, in a 3 mol l-1 guanidine solution, and also in sodium pectate and pectin solutions, b) of its high stability during a long-lasting storage at 4 °C, c) of its operational stability. The immobilization led to a partial change of the action pattern onto the high-molecular substrate, manifested in lowering the decrease of viscosity to degradation degree ratio.

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