Biomedical subjects

J Jozefonvicz

Publications and source records attributed to J Jozefonvicz.

At least 73 records · Page 4Linked to original sources

PubMed1991

Derivatized dextran inhibition of smooth muscle cell proliferation.

Proliferation of vascular smooth muscle cells is postulated to be one of the key events in the pathogenesis of atherosclerosis or during the development of focal glomerular sclerosis. Several studies have suggested that the antiproliferative effects of heparin appear to be regulated by different structural determinants. Our experiments show that dextrans substituted with carboxylic and benzylamide sulphonate groups markedly inhibit the growth of smooth muscle cells in vitro. Studies on the structure-function relationships of these products to their effect on rat aorta smooth muscle cells are reported. The antiproliferative capacity is similar to that of heparin.

Animals↗

PubMed1990

Double-coated silica supports for high-performance affinity chromatography of proteins.

In order to prepare easily derivatizable supports for high-performance affinity chromatography (HPAC), the advantages of traditional polysaccharide-based supports were combined with the excellent mechanical properties of silica by coating the porous silica beads with a double layer of polysaccharide. The starting material was preliminarily impregnated with dextran or agarose, substituted with a calculated amount of positively charged diethylaminoethyl (DEAE) functions, in order to neutralize the cation-exchange capacity. These silica beads were then recoated by a second coupling with a native dextran or agarose so the DEAE functions introduced by the first coating could be overlayed and the coating state of the silica beads was further improved. The passivation of silica was confirmed by eluting standard proteins on the double-coated silica supports in high-performance size-exclusion chromatography. The elution of an acidic biopolymer, heparin, on different coated silica supports under gradient conditions demonstrated the major improvement of the native polymeric overlayer on the ionic properties of the support. These double-coated silica supports can also easily be activated by classical activation methods and coupled with active ligands (protein A and heparin). The active supports grafted by protein A were used in HPAC of human immunoglobulin G. The double-coated silica supports grafted by heparin were used in HPAC of human alpha-thrombin and human antithrombin III and for the purification of bovine thrombin from a commercial crude thrombin preparation.

Animals↗

PubMed1990

Specific adsorption of serine proteases on coated silica beads substituted with amidine derivatives.

Amidine derivatives interact with serine proteases, the inhibition being due to interactions between amidine functions and the active sites of the enzymes. Five different types of amidine (substituted or unsubstituted) were coupled to coated silica beads, which had previously been coated with DEAE-dextran to minimize the non-specific interactions due to silanol groups. Coated silica functionalized with substituted amidines shows a strong affinity towards human plasmin. This affinity is probably due to hydrophobic interactions between the substituted amidine and the human plasmin structure. Coated silica grafted by p-aminobenzamide gives a specific interaction with human plasmin. The importance of ionic strength and the steric conformation of the ligand is discussed. This support was used to purify thrombin from crude preparations by high-performance affinity chromatography.

Adsorption↗

PubMed1990

High-performance affinity chromatography of basic fibroblast growth factor on polystyrene sulphonate resins modified with serine.

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor, so it was usually purified by affinity chromatography on a heparin-grafted support. It was previously observed that, among several modified polystyrene that exhibit anticoagulant heparin-like properties, insoluble polystyrenes modified by chlorosulphonation substituted with serine (PSSer) develop specific interactions with bFGF in solution. These PSSer resins were used as stationary phases in high-performance affinity chromatography in order to separate the radiolabelled growth factor from a bovine brain crude extract. The growth factor is strongly bound to the solid phase, as demonstrated by the adsorption isotherms. It can be adsorbed on the resin at low ionic strength and be desorbed by raising the salt concentration in the eluent. The effects of flow-rate, of the initial buffer and of the slope of the salt gradient on the adsorption and on the desorption of bFGF were investigated in order to study the thermodynamic and the kinetic parameters of the interactions and to define the optimum conditions for a fast and efficient separation of the growth factor.

Animals↗

PubMed1990

Affinity chromatography of fibroblast growth factors on substituted polystyrene.

The heparin-binding growth factors aFGF and bFGF (acidic and basic fibroblast growth factor) from crude bovine brain extract were co-eluted with purified [125I]aFGF and/or [125I]bFGF as tracers from heparin-Sepharose and from several insoluble substituted polystyrenes used as stationary phases in low-pressure affinity chromatography. The ability of the resins to isolate FGFs was determined by measuring the eluted radioactivity. It was demonstrated that the various substituted polystyrene resins retain [125I]aFGF and [125I]bFGF with different specificities according to the chemical nature of the substituted groups bound to the polystyrene support. Bifunctional resins substituted with sulphonate and phenylalanine sulphamide groups adsorbed both [125I]aFGF and [125I]bFGF whereas bifunctional resins substituted with sulphonate and sulphamide serine adsorbed only [125I]bFGF. These stationary phases could be adapted to high-performance affinity chromatography and used to isolate growth factors of the FGF family.

Animals↗

PubMed1990

Interactions of biospecific functional polymers with blood proteins and cells.

Biospecific functional polymers, i.e. synthetic or artificial polymers substituted with specific chemical functional groups carried by the macromolecular chain are designed to interact with living systems. These polymers are either insoluble or soluble, derived from polystyrene and dextran. Polymers substituted with aryl sulfonate and carboxyl groups specifically interact with antithrombin III and serine-proteases involved in the coagulation of blood. As a consequence, these polymers possess heparin-like activity and are therefore of low thrombogenicity when exposed to flowing blood. Other functional polymers have been prepared in order to interact with various components of the immune system. Soluble and insoluble functional polymers in contact with cells can affect both cell proliferation and metabolism. Some functional polymers have the ability to inhibit or to stimulate cell growth while others can alter cell function without a change in growth characteristics. The functional polymers described have possible applications as plasma expanders, non-thrombogenic catheters, non-complement activating surfaces and other applications in oncology, biotechnology and immunochemistry.

Adsorption↗

PubMed1989

Coated silica supports for high-performance affinity chromatography of proteins.

Polymer-coated silica supports are potentially good stationary phases for high-performance affinity chromatographic separations of proteins. Silica beads have been coated with a polysaccharide (dextran or agarose), substituted by a calculated amount of positively charged diethylaminoethyl functions in order to neutralize the negatively charged silanol groups of silica and to facilitate the formation of a hydrophilic polymeric layer on the inorganic surface. The silica-based supports were prepared in two steps. First, the silica was impregnated with a solution of diethylaminoethylated polymer, and then the coating polymer was crosslinked in order to avoid leakage of the polymeric layer. The supports present minimal non-specific interactions with proteins, as tested by high-performance size-exclusion chromatography. These coated silica supports were coupled with active ligands, such as protein A, concanavalin A and heparin, by conventional coupling methods. The resulting affinity stationary phases were tested by the elution of proteins in order to study their performance in high-performance affinity chromatography.

Antithrombin III↗

PubMed1989

New natural polysaccharides with potent antithrombic activity: fucans from brown algae.

Fucans extracted from brown algae exhibit anticoagulant properties, as demonstrated by coagulation assays and kinetic study of the inactivation of thrombin in the presence of antithrombin III. These derivatives exert a direct antithrombic activity by an antithrombin III-independent pathway. Besides, their anticoagulant activity is not mediated by a potency to inhibit factor Xa. Relatively low-molecular-mass fractions (50,000 Daltons) prepared by chromatographic fractionation of the crude fucans also exhibit anticoagulant activity. They may prove useful as anticoagulant drugs.

Antithrombin III↗

PubMed1988

Dextran-coated silica packings for high-performance size-exclusion chromatography of proteins.

Porous silica beads have excellent mechanical properties for the high-performance liquid chromatography of proteins. However, the importance of non-specific interactions between silanols on the silica surface and the proteins requires a modification of these supports before they can be used as stationary phases for size-exclusion chromatography (SEC). Silica beads were coated with dextran bearing a small number of positive charges in order to neutralize the negatively charged silanol groups. Diethylaminoethyl-dextrans (DEAE-dextrans) with a relatively low percentage of dextran units bearing DEAE functions were layered on the silica beads. The influence of several characteristics of these packings on chromatographic performance were studied in order to determine the optimal conditions for the SEC of proteins.

Chromatography, DEAE-Cellulose↗

PubMed1988

Thrombin binding properties of insoluble modified polystyrene: Part II.

Antithrombin III (AT III) inhibits thrombin via an arginine-serine interaction. Insoluble polystyrene resins grafted with arginyl methyl ester have been synthesized, and their interaction with thrombin tested. One of these resins was selected for its high affinity for thrombin. In this paper we report the characteristics of this thrombin resin interaction. Using this substituted polystyrene resin as a support for affinity chromatography, we have compared the binding of thrombin with that of other proteins (prothrombin, Factor IXa, trypsin and AT III). It was found that 0.7 mg of highly purified human thrombin (2,100 U/mg) was bound to 1 g of resin. This could only be eluted at high ionic strength (1.5 M) and the amidolytic and clotting activities of the eluted thrombin remained unchanged. The binding of thrombin to the resin involves the active site of the enzyme but also other residues since, when DIP thrombin was used, the inactive enzyme could be eluted at lower ionic strength (1.0 M). This resin seems to be specific for thrombin because it does not bind the other serine-proteases (trypsin or Factor IXa), prothrombin (the inactive precursor of thrombin) or AT III. The arginyl residues of the resin are important for the specificity of the interaction with Factor IIa since prolyl residues are totally ineffective. Chromatography performed on such a resin is a very efficient method of purifying thrombin, and may be very useful for the removal of thrombin as a contaminant of plasma protein fractions.

Antithrombin III↗

PubMed1988

Adsorption of thrombin from buffer and modified plasma to polystyrene resins containing sulphonate and sulphamide arginyl methyl ester groups.

The adsorption of albumin and thrombin to insoluble modified polystyrene resins bearing sulphonate (PSSO3) and L-arginyl methyl ester groups (PAOM) was investigated in both purified and plasma systems. Radioiodinated proteins were used to follow adsorption in a 'minicolumn' experiment. Albumin adsorption was found to follow the Langmuir model and specific surface areas of the various resins were evaluated from plateau albumin adsorption data. The adsorption isotherms of thrombin both in buffer and in antithrombin III/fibrinogen-free plasma were also found to be Langmuir-like, and the quantities adsorbed at the isotherm plateaux are in the monolayer range. Analysis of the isotherms at 4 degrees and 37 degrees C for the purified system shows that adsorption is endothermic. Adsorption capacities in plasma remain high (30-50% of those in the purified system) despite competition from the other plasma proteins. These data confirm the strong affinity and selectivity of these resins for thrombin.

Adsorption↗

PubMed1987

Affinity of antithrombin III for insoluble modified polystyrene.

In previous papers we described insoluble polystyrenes grafted with L-arginyl methyl ester (PAOM) which exhibit a high affinity for thrombin. Based on their specific interaction with the protease, the resin has been used as stationary phases for affinity chromatographic procedure of thrombin in affinity chromatography (AC) and high performance liquid affinity chromatography (HPLAC). A slight adsorption of antithrombin III (AT III) was shown in AC. In this paper we study the interaction of AT III for PAOM resin in batch procedure. The adsorption is measured using purified protein and corresponds to a monolayer adsorbed on the surface of the polymer. The affinity constant is evaluated kAT = 3.10(5) 1.M-1. Furthermore, insoluble sulfonated polystyrenes grafted with different amino acid were shown to possess an heparin-like behaviour and catalyse the generation of thrombin-AT III complex. So, compared to one of the resin previously mentioned, it could be concluded that PAOM is not heparin-like. When thrombin is adsorbed on the surface of the polymer, AT III cannot interact with the specific seryl residue of the enzyme.

Adsorption↗

PubMed1987

Anticoagulant hydrogels derived from crosslinked dextran. Part I: Synthesis, characterization and antithrombic activity.

Hydrogels have been prepared by binding various amino acids to Sephadex derivatives bearing carboxymethyl and sulphonated benzylamide groups. Depending on the chemical nature and content of the amino acid substituent, these insoluble strongly hydrophilic materials may absorb 7 to 19 volumes of buffer per volume of dry material. These hydrogels present antithrombic activity in relation to their swelling ratio. The adsorption of thrombin might partially explain the anticoagulant effect as shown by the evaluated affinity constants between the hydrogels and the enzyme i.e. 1 to 2 X 10(6) I/M.

Anticoagulants↗

PubMed1987

Anticoagulant hydrogels derived from crosslinked dextran. Part II: Mechanism of thrombin inactivation.

Sephadex derivatives bearing carboxymethyl, sulphonated benzylamine and amino acid groups exhibit heparin-like behaviour as demonstrated by the kinetic study of the thrombin inactivation in the presence of antithrombin III. Furthermore, whatever the chemical composition of these hydrogels, the diffusion coefficient of thrombin remained approximately constant and could not be connected with the variation of the antithrombin activity of the resins. Hence, in the heparin-like mechanism, the diffusion rate of thrombin inside the beads of hydrogels was not the limiting step. In fact, the swelling ratio (varying according to the chemical composition of these biomaterials) was involved in the anticoagulant properties of the resins.

Anticoagulants↗

PubMed1987

Molecular weight dependency of the acquired anticomplementary and anticoagulant activities of specifically substituted dextrans.

Dextran that had been substituted with carboxylic and benzylamine sulphonated groups was fractionated by gel chromatography into fractions, of narrow molecular weight distribution from 6000 to 190,000 daltons and of similar chemical composition. The fractions exhibited anticomplementary and anticoagulant activities that rapidly increased with molecular weight and tended to plateau above approximately 20,000 and 40,000 daltons respectively. Anticoagulant activity was lower than that of heparin, whereas the capacity of the fractions to inhibit formation of the classical and alternative C3 convertases in a purified system was similar to that of heparin and their ability to inhibit CH50 in whole serum was higher than that of heparin. The data argue for a random distribution of structurally independent anticoagulant and anticomplementary sites along the macromolecular chains of substituted dextrans.

Anticoagulants↗

PubMed1986

Thrombin purification by one-step preparative affinity chromatography on modified polystyrenes.

Insoluble polystyrenes substituted with sulphonate and L-arginyl methyl ester have been synthesized. Using their specific affinity for thrombin, we developed a simple one-step chromatographic procedure for thrombin purification. As a control, insoluble polystyrenes substituted only with sulphonate groups were tested. The results obtained confirmed the importance of the arginyl residues grafted onto these polymers to obtain an affinity matrix useful for purifying thrombin with a high specific activity and a good recovery.

Chromatography, Affinity↗

PubMed1986

High-performance affinity chromatography of human thrombin on modified polystyrene resins.

Cross-linked polystyrenes modified with L-arginyl methyl ester mimic the binding site of antithrombin III and thrombin substrates. They can be used as stationary phases in high-performance affinity chromatography of thrombin. Under isocratic conditions, thrombin is strongly adsorbed on the resins when the sodium chloride concentration is lower than 0.5 M. The bound enzyme can be selectively desorbed when the salt concentration is raised to about 1.2 M. With a linear salt gradient, the specific elution of thrombin can be effected with a high recovery of its enzymatic activity. The decomposition products of thrombin, when treated with sodium dodecyl sulphate, are not retained by the stationary phase. The effects of the flow-rate and salt gradient slope on the adsorption and desorption of alpha-thrombin demonstrate the importance of kinetic parameters.

Adsorption↗

PubMed1986

Affinity chromatography of thrombin on modified polystyrene resins.

Insoluble polystyrenes substituted with sulphonate and L-arginyl methyl ester (PAOM) present substituents mimicking the reactive binding site of antithrombin III. These materials have a specific affinity for thrombin. The binding of the enzyme is reversible and the eluted thrombin remains active. Consequently, these resins can be used as stationary phases in affinity liquid chromatography in order to purify thrombin with a high biological activity. The influence of different characteristics of such polymers (substitution ratio, average particle size, affinity constant, synthesis conditions) on the purification performance is studied. Human prothrombin complex concentrate is activated and applied onto the gel. A purified human thrombin of high specific activity is separated with a high recovery of biological activity of the enzyme.

Chromatography, Affinity↗