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J Jozefonvicz

Publications and source records attributed to J Jozefonvicz.

At least 55 records · Page 3Linked to original sources
PubMed1995

High-performance affinity chromatography of insulin on coated silica grafted with sialic acid.

High-performance liquid affinity chromatography (HPLAC) is a powerful method for the purification of biological compounds, owing to its specificity, speed and high resolution. We developed new chromatographic supports based on porous silica beads. In order to minimize non-specific interaction between the silanol groups at the silica surface and biological molecules, the beads are coated with dextran carrying a calculated amount of positively charged functions. Such supports have the mechanical properties of the starting inorganic material. Moreover, they can be easily activated and functionalized by active ligands using conventional coupling methods. In the present study, N-acetylneuraminic acid (NANA), a member of the sialic acid family, is coupled to dextran coated silica beads to obtain affinity supports. This class of compounds seems to play an important role in the cell recognition mechanism. In particular, sialic acids are present in the structure of the cellular receptors for insulin. By HPLAC, we can study the interactions between coated silica grafted with NANA and insulin. It is also possible to use these active supports to purify the compounds by affinity chromatography. However, it is important to determine and optimize the conditions for adsorption and desorption of insulin on supports grafted with sialic acid and to estimate the chromatographic performances of these active phases.

Amino Acid Sequence↗
PubMed1995

Effect of substituted dextran derivative on complement activation in vivo.

A soluble dextran derivatized with carboxylic groups (73%) and benzylamide sulphonate groups (15%), termed CMDBS 25, exhibited significant anticomplementary activity in the absence of anticoagulant activity. The polysaccharide inhibited both classical and alternative pathway-dependent complement activation in human and rat serum in vitro. Simultaneous administration of CMDBS 25 (100 mg) and crushed Sephadex G25 (20 mg) into normal Lewis rats suppressed systematic complement consumption that was induced by Sephadex in the animals by 98% for 1 h. Two consecutive injections of 100 mg of CMDBS at 1 h interval resulted in total suppression of systemic complement activation for 2 h and in 50% suppression for an additional 2 h. Infusion of CMDBS alone was well tolerated and had no effect on CH50 in serum in vivo. Our results demonstrate that CMDBS 25 exhibits anticomplementary properties in vivo and suggest that the polymer represents a potential therapeutic agent for pathological conditions associated with complement activation.

Animals↗
PubMed1994

Inhibition of complement activation by natural sulfated polysaccharides (fucans) from brown seaweed.

In the present study, we demonstrate that natural sulfated polysaccharides (fucans) isolated from brown seaweed are potent inhibitors of human complement activation. A fucan fraction of chromatographic molecular weight 22,600, termed BS8, was found to inhibit classical and alternative pathway activation in whole serum in a dose-dependent fashion. Fucan BS8 inhibited formation of the classical pathway C3 convertase by interfering with C1 activation or by inhibiting C4 cleavage and the interaction between C4b and C2. The fucan also inhibited formation/function of the alternative pathway C3 convertase by suppressing the binding of B to C3b and by interfering with the stabilizing function of Properdin. The inhibitory effect of fucans on formation of the C3 convertases was dependent on the molecular weight of the polysaccharide for compounds of chromatographic molecular weight below 16,600. Fucan had no effect on the function of the terminal complex. Since fucans were more efficient than heparin in inhibiting activation of the classical pathway in whole serum and exhibited a lesser specific anticoagulant activity on a molar basis, our results suggest that these natural sulfated polysaccharides have a potential for use as anti-complementary and anti-inflammatory agents.

Complement Activation↗
PubMed1994

Effect of a derivatized dextran on human osteoblast growth and phenotype expression.

Water soluble derivatized dextran named E9 with a molecular weight of 45,000 g l-1 containing 58% methyl carboxylic acid unit, 19% benzylamide unit, and 26% sulfonate with a specific anticoagulant activity of 0.29 IU mg-1 was studied for its effects on human osteoblast growth and phenotype expression for short-term treatment. At concentrations between 1 ng ml-1 and 1 microgram ml-1 E9 has no effect on DNA synthesis whereas at higher concentrations DNA synthesis is inhibited in a dose related fashion (87% for 400 micrograms ml-1). For concentrations which do not modify osteoblast growth, E9 promotes alkaline phosphatase activity, type I collagen and osteocalcin synthesis with a maximum effect for 0.1-1 microgram ml-1. It has a synergistic effect with hPTH increasing AMPc. Moreover, osteonectin synthesis was enhanced in a dose-dependent manner between 0.1 and 5 micrograms ml-1. These results seem to indicate that E9 is able to stimulate human osteoblast phenotype expression and could be useful in clinical applications.

Adult↗
PubMed1994

Tyrosyl sulfamide derivatives as ligands for affinity adsorption of anti FVIII antibodies.

Human anti factor VIII (anti FVIII) antibodies neutralize factor VIII procoagulant activity. These antibodies appear in about 5-10% of severely affected haemophiliac A patients treated with FVIII concentrates. In order to obtain non-thrombogenic materials able to interact specifically with anti FVIII, we have grafted amino acid residues which mimic part of the epitope of the FVIII molecule recognized by the anti FVIII. For this purpose, crosslinked polystyrenes functionalized with sulfonate and tyrosyl or methyl ester tyrosyl sulfamide groups have been synthesized and characterized. The in vitro removal of anti FVIII from haemophiliac patient plasma with antibodies, was performed on these different active supports. These polymers exhibit strong and selective affinity for the anti FVIII. The adsorption of the antibodies vary with the percentage of units bearing methyl ester tyrosyl sulfamide groups and present a maximum at 25% grafting rate. For the more efficient resin, the affinity constant, determined for the adsorption isotherm for the anti FVIII is about 10(9) M-1, whereas the affinity constant for the IgG in the same experimental conditions, is low (10(5) M-1). The influence of different chemical groups on the polymeric phase, on their affinity for the inhibitors was also studied. The most active resins can be selected and used in an extracorporeal circulation to reduce the concentration of anti FVIII in a blood epuration process.

Adsorption↗
PubMed1993

Affinity chromatography of sulphated polysaccharides separately fractionated on antithrombin III and heparin cofactor II immobilized on concanavalin A--sepharose.

Three sulphated polysaccharides, dermatan sulphate, fucan and heparin, were fractionated according to their affinity towards antithrombin III (ATIII) and heparin cofactor II (HCII), the two main physiological thrombin (IIa) inhibitors. Both inhibitors were immobilized on concanavalin A-Sepharose, which binds to the glycosylated chains of the proteins while the protein-binding site for the polysaccharide remains free. Each polysaccharide was fractionated into bound and unbound fractions either for ATIII or HCII. The eluted fractions were tested for their ability to catalyse ATIII/IIa and HCII/IIa interactions. The possible presence of a unique binding site for ATIII and HCII, on each sulphated polysaccharide, was also studied.

Animals↗
PubMed1993

In vitro stimulation of human endothelial cells by derivatized dextrans.

Derivatized dextrans exert a stimulatory effect on the in vitro growth of human umbilical vein endothelial cells (HUVEC). Measurements of growth were monitored by [3H]thymidine uptake and cell numbers. Our results show that some derivatized dextrans at 4 micrograms/ml (88 nM) increase the [3H]thymidine incorporation, whereas starting dextran (40,000 Da), dextran sulfate, and carboxymethyl dextran have no effect. In addition, heparin under similar experimental conditions shows a slight inhibitory effect on the HUVEC growth. The stimulatory effect of derivatized dextrans was also found when HUVEC grew during 7 days in medium containing 2% fetal bovine serum. We also observed that derivatized dextrans had no effect on the mitogenic activity of acidic fibroblast growth factor, a mitogenic factor for several cell types including HUVEC. By assessment of [3H]thymidine uptake at 48 h without serum, we concluded that the exogenous growth factors were not involved in the proliferative activity of these components. The stimulatory effects are related to the chemical nature and the proportion of substituents on the synthetic polysaccharides. The data indicate that benzylamide sulfonated groups play a key role in the stimulation of HUVEC growth. Neither carboxyl nor sulfate groups alone exhibit this effect. Thus, the stimulatory capacity of dextran derivatives depends strongly on the respective ratios of the functional groups.

Carbohydrate Sequence↗
PubMed1993

Antiproliferative capacity of synthetic dextrans on smooth muscle cell growth: the model of derivatized dextrans as heparin-like polymers.

Proliferation of vascular smooth muscle cells (SMC) is postulated to be a key step in the pathogenesis of atherosclerosis or restenosis after vascular interventions such as angioplasty. Natural glycosaminoglycans, such as heparin and heparan sulfate, are known for their ability to inhibit SMC proliferation in vivo and in vitro. The antiproliferative activity of synthetic derivatized dextrans exhibiting heparin-like anticoagulant and anticomplement capacities have been investigated with rat aorta smooth muscle cells in culture. We report here that some derivatized dextrans grafted with benzylamide sulfonate moieties are potent antiproliferative agents for rat smooth muscle cell (SMC) in vitro. These synthetic polymers inhibit the SMC proliferation as well as heparin. The SMC growth inhibition is dose dependent, reversible and non-toxic. Highly anionic carboxylic dextrans are not capable of inhibiting the SMC growth, excluding a simple charge effect mechanism. Using fluorescent (DTAF) probes, we demonstrated that the synthetic antiproliferative polymers and heparin are internalized into the SMC. No binding or internalization was observed with native dextran devoid of antiproliferative capacity. We conclude that a suitable distribution of functional groups on the dextran backbone can simulate heparin activity in terms of antiproliferative capacity on SMC growth.

Animals↗
PubMed1992

Derivatized dextrans mimic heparin as stabilizers, potentiators, and protectors of acidic or basic FGF.

Acidic and basic fibroblast growth factors (aFGF and bFGF) belong to a family of structurally related polypeptides characterized by a high affinity for heparin. a and bFGF display mitogenic activity for many cell types. Biological activity is strongly potentiated by heparin which stabilizes their molecular conformation by preventing physicochemical or enzymatic degradation. In our previous study we have shown that a water-soluble derivatized dextran named DDE, containing 82.2% methyl carboxylic acid groups, 6.1% benzylamide, and 5.6% sulfonate with a specific anticoagulant activity equivalent to heparin of 0.5 IU/mg could potentiate the mitogenic activity of aFGF on CCL39 cells. Optimal concentrations for maximal potentiation of 400 micrograms/ml and 20 micrograms/ml were obtained respectively for DDE and heparin. In the present report, we have uncovered the fact that several carboxymethyl benzylamide sulfonate dextrans differing in degree and positioning of the substituent groups can mimic heparin in regard to the protection, stabilization, and potentiating effects with aFGF or bFGF. Our data establishes that the dextran derivatives studied can act as potentiating agents for FGFs. Native dextran (DDA) had no effect. Dextran derivatives can also protect aFGF and bFGF from heat as well as from pH denaturation, and against trypsic and chymotrypsic degradation. The dextran derivative DDI (82% methylcarboxylic acid, 23% benzylamide, 13% sulfonate) was studied in greater detail and exhibited a greater protection for bFGF and a lesser protecting effect for aFGF than heparin. Derivatized dextrans which have very weak anticoagulant activity are of great interest as alternatives to heparin for use as stabilizers, potentiators, protectants, and slow-release matrices for FGFs in pharmaceutical formulations.

Animals↗
PubMed1992

Inhibitory effect of substituted dextrans on MCF7 human breast cancer cell growth in vitro.

Substituted dextrans can reproduce some of the properties of heparin and can thus be used to alter cellular growth. We studied the effect of heparin (H108), dextran (D), carboxymethylbenzylamide dextran (CMDB) and carboxymethylbenzylamide sulfonate dextran (CMDBS) on the growth of human mammary cells of the MCF7 tumor line. The cells were cultured in minimum Eagle's medium containing 2% fetal calf serum without biopolymer, or with increasing concentrations of H108, D, CMDB or CMDBS. Growth curves were accurately based on cell counting using a Coulter counter. Cell distribution in the various phases of the cycle was analyzed by flow cytometry. Dose-dependent growth inhibitory effects (400-4000 micrograms/ml) were observed. The effect on MCF7 tumor cells was most apparent with CMDBS. The percentage of cells in the S phase decreased with preferential blocking in the G0/G1 phase. Pre-clinical studies can be anticipated as there is an absence of in vivo toxicity.

Aged↗
PubMed1992

Inhibition by heparin and derivatized dextrans of Staphylococcus aureus adhesion to fibronectin-coated biomaterials.

Recent data on cardiovascular device-centered infections suggest that some plasma and extracellular matrix proteins contribute to bacterial adhesion and colonization on biomaterials. We previously developed an in vitro assay to study the Staphylococcus aureus adhesion-promoting effect of surface-adsorbed fibronectin on flat PMMA coverslips coated with a monolayer amount of fibronectin. We screened the potential anti-adhesive properties of a group of substituted dextrans, previously shown to exhibit potent anticoagulant and anticomplementary activities. In comparison to unsubstituted dextran which showed no significant (< 20%) adhesion inhibition at 1 mg/ml, dextrans increasingly substituted with carboxylic and benzylamide groups (CMBD) exhibited increasing anti-adhesive activities. Three CMBD derivatives showing an increasing proportion (5-14%) of benzylamide groups showed inhibition of bacterial adhesion increasing from 33 to 51% at 1 mg/ml. Another category of substituted dextrans having a variable proportion (2-26%) of sulfonated benzylamide groups (CMBDS) produced active inhibition of S. aureus adhesion. In comparison to these heparin-like dextran derivatives, native heparin produced inhibition values of S. aureus adhesion which were intermediate between those of CMBD and CMBDS compounds. Furthermore, the anti-adhesive activity was still expressed when substituted dextrans were preincubated with fibronectin-coated PMMA but washed away at the time when radiolabeled bacteria were added to the adhesion assay. This indicates that the anti-adhesive effects of CMBDS could be exerted at the level of the S. aureus binding site of fibronectin. In conclusion, S. aureus adhesion on fibronectin-coated biomaterials can be efficiently blocked in vitro by soluble compounds such as dextran derivatives.

Adsorption↗
PubMed1991

Anticoagulant properties of a fucoïdan fraction.

Fucoïdans are a family of high molecular weight sulphated polysaccharides in the Mr range 8 x 10(5) -10(6), widely dispersed in brown seaweed cell wall. When extracted from several brown algae, they exhibit anticoagulant properties. The chemical degradation of a crude extract, from Pelvetia canaliculata, was undertaken to obtain a low molecular weight polysaccharide (Mr 20,000 +/- 5,000) with the purpose of a possible clinical use. Its anticoagulant potency was investigated through the inhibition of factor IIa and factor Xa in the presence of antithrombin III or heparin cofactor II. The degraded fucoïdan revealed a potent antithrombin activity: studied in an antithrombin III depleted plasma or in the presence of purified heparin cofactor II, the fucoïdan was as efficient as heparin and dermatan sulphate on heparin cofactor II potentiation, at the same concentration by weight. In whole plasma or in the presence of the purified inhibitor, an anti-factor IIa activity mediated by antithrombin III was detected (30 times less potent than for heparin, on a weight to weight basis). In contrast, no anti-factor Xa activity was detected in the presence of the degraded fucoïdan, under the same experimental conditions. These fucoïdans, by-products of alginates preparation in the food and cosmetologic industries, are obtained easily. Thus, they may represent a cheap and easy source of a new type of anticoagulants.

Anticoagulants↗
PubMed1991

Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports.

The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non-specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared.

Adsorption↗
PubMed1991

Immobilization of dermatan sulphate on a silica matrix and its possible use as an affinity chromatography support for heparin cofactor II purification.

Dermatan sulphate (DS) is a glycosaminoglycan which catalyses specifically thrombin inhibition by a plasmatic inhibitor, Heparin cofactor II (HCII). DS was insolubilized on a silica matrix to study its interaction with HCII. The immobilization of DS was performed with a good yield on a silica previously coated with polysaccharides in order to neutralize the negatively charged silanol groups. The value of the affinity constant of insolubilized DS for HCII, measured by the adsorption isotherm, is consistent with the value obtained for soluble DS. The DS bound to the silica matrix was also tested as a chromatographic support for the purification of HCII from human plasma; the optimum conditions for HCII adsorption and desorption were determined. The eluted HCII was obtained with a good yield (21%) and with no contamination by antithrombin III, the other main plasmatic inhibitor of thrombin.

Chromatography, Affinity↗
PubMed1991

Affinity chromatography of fibroblast growth factors on coated silica supports grafted with heparin.

Dextran-coated silica beads are excellent supports for affinity chromatography of proteins. They can be easily grafted using conventional coupling methods with different active ligands, such as heparin. Fibroblast growth factors develop specific interactions with heparin through well-defined amino acids sequences. The heparin-dextran coated silica phases exhibit an affinity for these growth factors. Under our experimental conditions, the basic form can be absorbed on the solid support at a moderate salt concentration (0.5 M sodium chloride) and can be selectively desorbed by increasing the ionic strength of the eluent. The purification performances of such phases are compared to those obtained on the heparin grafted soft gels. Because of their mechanical properties, the dextran-coated silica supports were also used in high-performance affinity chromatography to purify fibroblast growth factors from a bovine brain crude extract.

Animals↗
PubMed1991

Interactions of proteins in human plasma with modified polystyrene resins.

Investigations are reported on the composition of protein layers adsorbed from plasma to various modified polystyrene resins. As well as polystyrene itself, polystyrene bearing sulfonate groups in the benzene rings, and polystyrene sulfonate in which the sulfonate groups were converted to amino acid sulfamide, were investigated. Some of these resins were shown in previous work to have anticoagulant properties. To study the adsorption of proteins from plasma, the resins were exposed to citrate anticoagulated human plasma for 3 h. Adsorbed proteins were then eluted sequentially by 1M Tris buffer and 4% SDS solution, and examined by SDS-PAGE. The gel patterns were similar on all resins except polystyrene. From the MWs of the gel bands, the major protein component appeared to be fibrinogen. Smaller amounts of plasminogen, transferrin, albumin, and IgG were also present. In addition, Ouchterlony immunoassay of the eluates from one resin gave positive identification of complement C3, fibronectin, IgG, and IgM. Many other minor gel bands remain unidentified. A consistent finding for all resins was the presence of plasmin-type fibrinogen degradation products though the amounts varied with resin type. It is concluded from this (and from experiments showing FDP formation when fibrinogen was absorbed to the resins, from buffer containing a trace of plasminogen) that the functional groups in these materials promote the adsorption of plasminogen and its activation to a plasmin-like molecule. It appears from the substantial quantities of fibrinogen adsorbed to these materials after 3 h exposure to plasma that the Vroman effect (giving transient adsorption of fibrinogen) is not operative on these materials. It is hypothesized that specific interactions occur between fibrinogen and sulfonate groups.

Adsorption↗
PubMed1991

Enzymatic degradation and immunogenic properties of derivatized dextrans.

Dextran T40 was modified with carboxylic, benzylamide and benzylamine-sulphonated groups (samples 1-10). The polymers were incubated with dextranase and the decrease of molecular weight was determined by high performance size exclusion chromatography. It was shown that the higher the substitution of dextrans, the lower their degradability. Modification of dextran with benzylamine and benzylamine-sulphonated groups appeared to hinder the formation of the enzyme-substrate complex more than the same quantity of carboxylic groups. The immunogenicity of one of the modified dextrans, containing 54% of carboxylic groups and 19.5% of benzylamine-sulphonated groups, was determined after subcutaneous and intravenous administration into Balb/c mice. The antibody titre was very low even if administered in complete Freund's adjuvant and did not depend on the injected dose. On the average, the titres of antibodies were lower by five orders of magnitude compared to bovine gamma globulin.

Animals↗