DIFFERENTIAL STABILITY OF IODINE POOR AND IODINE RICH RAT THYROGLOBULIN

1974 ◽  
Vol 75 (1) ◽  
pp. 33-49 ◽  
Author(s):  
Lubomir J. Valenta
Keyword(s):  
Ionic Strength ◽  
Succinic Anhydride ◽  
Iodine Concentration ◽  
Freezing And Thawing ◽  
Sedimentation Pattern ◽  
Differential Stability ◽  
Chaotropic Anions ◽  
Low Ionic Strength ◽  
Covalent Interactions

ABSTRACT In certain environments thyroglobulin is dissociated into 12S subunit or unfolded into 14–17S species. The unfolding and dissociating ability of various agents was examined on poorly iodinated (PIT) or iodine rich (IRT) rat thyroglobulins. According to the efficiency of provoking unfolding or dissociation these agents could be grouped in the following order: low ionic strength < chaotropic anions < chaotropic anions plus freezing and thawing < succinic anhydride and mild alkali < SDS < strong alkali. The degree of thyroglobulin changes was further dependent on the iodine concentration. No change of sedimentation pattern was obtained in the presence of 2-mercaptoethanol or after incomplete reduction and alkylation of the iodine rich protein. No stabilization of PIT was brought about by a number of oxidizing agents. Stabilization was only obtained by in vitro iodination. The main difference between the uneffected and unfolded or dissociated thyroglobulin species was in the amount of thyroxine, or, at lower levels of iodination, DIT. The results suggest that non-covalent, especially hydrophobic, rather than covalent interactions are involved in stabilization of conformation of the thyroglobulin molecule.

Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method

1981 ◽  
Author(s):  
R Jordan ◽  
T Zuffi ◽  
M Fournel ◽  
D Schroeder
Keyword(s):  
Ionic Strength ◽  
High Activity ◽  
Tight Binding ◽  
Therapeutic Benefit ◽  
Purification Scheme ◽  
The Matrix ◽  
Low Ionic Strength ◽  
Potential Therapeutic Benefit

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro.

1988 ◽  
Vol 107 (5) ◽  
pp. 1793-1797 ◽  
Author(s):  
W S Sale ◽  
L A Fox
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Glass Surface ◽  
Functional Assay ◽  
Atpase Subunits ◽  
Low Ionic Strength ◽  
Intermediate Chains ◽  
Sea Urchin Sperm

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.

scholarly journals Accessibility of epitopes on fibrin clots and fibrinogen gels

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1469-1475 ◽  
Author(s):  
R Procyk ◽  
B Kudryk ◽  
S Callender ◽  
B Blomback
Keyword(s):  
Monoclonal Antibody ◽  
Clinical Trials ◽  
Ionic Strength ◽  
Factor Xiii ◽  
Imaging Agent ◽  
Low Ionic Strength ◽  
Clot Structure ◽  
Radiolabeled Antibodies ◽  
Fibrin Clots

Abstract Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241– 476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4–2/xl-f, anti gamma 392–406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15–21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1- reactive epitopes. An antibody that does not recognize fibrin (antibody 1–8C6, antiB beta 1–21) predictably did not bind to fibrin clots and bound to 35% of the 1–8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.

scholarly journals Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

1982 ◽  
Vol 95 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M S Mooseker ◽  
T D Pollard ◽  
K A Wharton
Keyword(s):  
Ionic Strength ◽  
Intestinal Epithelial ◽  
Capping Protein ◽  
The Core ◽  
Nucleated Polymerization ◽  
Myosin Subfragment ◽  
Brush Borders ◽  
Pointed End ◽  
Low Ionic Strength

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

scholarly journals VASP is a processive actin polymerase that requires monomeric actin for barbed end association

2010 ◽  
Vol 191 (3) ◽  
pp. 571-584 ◽  
Author(s):  
Scott D. Hansen ◽  
R. Dyche Mullins
Keyword(s):  
Ionic Strength ◽  
Total Internal Reflection ◽  
Molecular Mechanisms ◽  
Binding Modes ◽  
Cellular Functions ◽  
Actin Networks ◽  
Filament Assembly ◽  
Binding Event ◽  
Low Ionic Strength

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (Kd = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (Koff = 0.69 s−1) polymerases that deliver multiple actin monomers per barbed end–binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.

In vitro and in vivo evaluation of xanthan gum–succinic anhydride hydrogels for the ionic strength-sensitive release of antibacterial agents

2016 ◽  
Vol 4 (10) ◽  
pp. 1853-1861 ◽  
Author(s):  
Bailiang Wang ◽  
Yuemei Han ◽  
Quankui Lin ◽  
Huihua Liu ◽  
Chenghui Shen ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Xanthan Gum ◽  
Succinic Anhydride ◽  
Antibacterial Agents ◽  
Lower Degree ◽  
Infection Model ◽  
In Vivo Evaluation

XG–SA/GS hydrogels yielded a significantly lower degree of infection than native XG–SA hydrogels in an in vivo rabbit subcutaneous S. aureus infection model at day 7.

scholarly journals Accessibility of epitopes on fibrin clots and fibrinogen gels

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1469-1475 ◽  
Author(s):  
R Procyk ◽  
B Kudryk ◽  
S Callender ◽  
B Blomback
Keyword(s):  
Monoclonal Antibody ◽  
Clinical Trials ◽  
Ionic Strength ◽  
Factor Xiii ◽  
Imaging Agent ◽  
Low Ionic Strength ◽  
Clot Structure ◽  
Radiolabeled Antibodies ◽  
Fibrin Clots

Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241– 476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4–2/xl-f, anti gamma 392–406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15–21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1- reactive epitopes. An antibody that does not recognize fibrin (antibody 1–8C6, antiB beta 1–21) predictably did not bind to fibrin clots and bound to 35% of the 1–8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.

ROLE OF TAMM-HORSFALL PROTEIN (THP) IN LITHOGENIC PROCESS : ANIMAL STUDIES

2019 ◽  
pp. 1-2
Author(s):  
Varsha Choudhary
Keyword(s):  
Ionic Strength ◽  
Guinea Pigs ◽  
Animal Studies ◽  
Dual Role ◽  
Urinary Oxalate ◽  
Crystal Aggregation ◽  
Physico Chemical ◽  
Low Ionic Strength

One of the defenses against nephrolithiasis is provided by macromolecules that modulate the nucleation, growth, aggregation and retention of crystals in the kidneys.According to its well-known physico-chemical properties.THP has a dual role in modifying crystal aggregation: at high pH and low ionic strength (IS),THP is a powerful crystal aggregation inhibitor.Upon lowering pH and rasing ionic strength THP viscosity increases,leading to reduced crystal aggregation inhibition.For this purpose eight guinea pigs were made hyperoxaluric.The treatment was given for fifteen days;then urine samples were collected before treatment ;then on 5,10,15and 25 day( after treatment) and in vitro addition of THP on 30th day which was isolated from hyperoxaluric and normal animals.The effect of EG+GM on urinary oxalate,THP and TBAR levels increases but after treatment the urine chemistry revert to normal profile,though plasma TBAR levels were appreciably high.The crystallization of calcium was almost double when THP was isolated from hyperoxaluric animals rather than normals.Our study suggested that THP act as a promoter

scholarly journals ANTIMICROBIAL ACTIVITY OF LYSOZYME TOWARDS LISTERIA MONOCYTOGENES AT VARIOUS MEDIUM CONDITIONS

2019 ◽  
Vol 19 (1S) ◽  
pp. 161-162
Author(s):  
M N Berlov ◽  
S V Legkovoy ◽  
E S Umnyakova ◽  
V N Kokryakov
Keyword(s):  
Antimicrobial Activity ◽  
Listeria Monocytogenes ◽  
Ionic Strength ◽  
Bacterial Growth ◽  
Human Leukocyte ◽  
Antimicrobial Action ◽  
Positive Bacterium ◽  
Enzymatic Action ◽  
Low Ionic Strength

The in vitro antimicrobial action of human leukocyte lysozyme from on gram-positive bacterium Listeria monocytogenes under various medium conditions was studied. It was shown that in a low ionic strength buffer (without NaCl), lower doses of lysozyme are required to reveal the microbicidal effect than in the case of 0.075 or 0.15 M NaCl. The bacterial growth phase does not significantly affect the antimicrobial activity of lysozyme. The results obtained are consistent with the two-stage mechanism of the antimicrobial action of lysozyme, which includes enzymatic and non-enzymatic action.

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