Interactions of human hemoglobin with high-molecular-weight dextran sulfate and diethylaminoethyl dextran

The binding of hemoglobin with high molecular weight dextran sulfate and diethylaminoethyl (DEAE) dextran was studied by turbidimetry, viscosimetry, ultrafiltration and oxygen affinity measurements. The results indicate that a complex is formed between hemoglobin and the charged dextrans through salt bridges. This complexation can lead to precipitation if the ionization degree of hemoglobin is high enough to develop an extensive neutralization of the charges of polyionic dextrans. At the pH value near to the isoelectric point, complexation still occurs, probably through salt bridges between the polyelectrolytes and the ionized sites of opposite charge on the hemoglobin molecule, but the complexes remain soluble in solution. Binding appears to be stronger for the hemoglobin-dextran sulfate compounds than for the hemoglobin-DEAE dextran compounds. The oxygen affinity of hemoglobin is greatly decreased in the presence of dextran sulfate, indicating a stabilization of the deoxyhemoglobin with respect to the oxyhemoglobin.     

Synthesis and characterization of a polyoxyethylene derivative for the affinity labeling of human hemoglobin

The possibility of preparing a polyoxyethylene derivative containing only one active group per chain, and with a narrow distribution of molecular size, was investigated. The aim of this investigation was to obtain a polymeric derivative of benzenehexacarboxylate (BHC) capable of interacting specifically inside the polyphosphate binding site of human hemoglobin (Hb) and which, after covalent coupling with this protein, could lead to conjugates with well-defined oxygen-binding properties. Such conjugates could be of interest in the field of blood substitutes. To minimize the percentage of high-molecular-weight products resulting from the fact that, on one hand, commercial monomethoxypolyoxyethylene (MPOE 5000) contained a considerable amount of difunctional and high-molecular-weight species, and on the other hand that several reactions were likely to lead to cross-linked products, fractionation steps and/or chromatographic steps were carried out. The final compound obtained under these conditions (83,5% of BHC-mono substituted polyoxyethylene, 8,5% of BHC disubstituted and 8% MPOE) was found to interact with the polyphosphate binding site of Hb, as it was capable of lowering its affinity for oxygen. After covalent coupling with deoxy-Hb under defined conditions, this effect was strengthened, which means that the binding was probably formed with an amine of the Hb polyphosphate binding site.     

Thermosets obtained by chemical modification of poly(epichlorohydrin) with vinyl-terminated aromatic carboxylates

Poly(epichlorohydrin) was modified in a high extent with several vinyl-terminated aromatic potassium carboxylates (4-(2-propenyloxy)benzoate, 4-(5-hexenyloxy)benzoate, 3-(2-propenyloxy)-2-naphthoate, 3-(5-hexenyloxy)-2-naphthoate and 2-(thioallyl)nicotinate) under solid-liquid phase-transfer conditions. Simultaneously with this substitution reaction, cleavage and crosslinking side reactions occurred. The polymers obtained were characterized by NMR, IR, elemental analysis, viscosimetry, size exclusion chromatography (SEC), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Then, crosslinked materials were obtained by curing mixtures of these linear polymers with 10% w/w of a radical initiator (dicumyl peroxide or 2,2′-azoisobutyronitrile) on a DSC device. Finally, the thermal stability of the thermosetting materials was tested by TGA, and the Arrhenius kinetic parameters were determined for dicumyl peroxide initiated crosslinking processes by calorimetric measurements.     

Synthesis and characterization of biodegradable polylactide-grafted dextran and its application as compatilizer

Brush-like biodegradable polylactide-grafted dextran copolymer (PLA-g-dextran) was by a bulk polymerization reaction using a trimethylsilyl-protected (TMS) dextran as macroinitiator and stannous octoate as catalyst. After the polymerization, the TMS groups could be easily removed by immersing the copolymer in methanol for 48 h. The PLA-g-dextran copolymers were characterized by (1)H NMR, GPC and intrinsic viscosity measurements. Besides, mouse 3T3 fibroblasts were cultured on these copolymeric substrates together with pure polylactide (PLA). Although the copolymers exhibited better hydrophilicity and cell affinity compared to pure PLA because of the incorporation of glucose units and the brush-like architecture, it was found that the cells still could not migrate into the center part of scaffold made of PLA-g-dextran copolymer. In result, PLA-g-dextran copolymers themselves were not an appropriate choice for the cell scaffold material, however, it could be used as compatilizer to ameliorate the compatibility between hydrophilic dextran and hydrophobic PLA due to its amphiphilic structure, which could improve the mechanical properties of PLA/dextran blends by reducing the phase separation between PLA and dextran. Therefore, the PLA/dextran blends, which had good cell affinity and moderate mechanical strength, might be prospect cell scaffold materials.     

Anticoagulant activity of dextran derivatives. Part I: Synthesis and characterization

Substituted dextrans bearing carboxymethyl and benzylsulphonate groups have been prepared. These materials exhibit an antithrombic activity correlated with the ratio of each substituent. The highest activity is obtained when the dextran derivative contains more than 50% of carboxylic acid groups and simultaneously about 15% of benzylsulphonate functions.     

Production and characterization of antibodies specific for domains of human IgM

A method of preparing antibodies against cμ3 and cμ4 domains of human IgM is described. cμ3- and cμ4-binding antibody fractions were isolated by affinity chromatography from IgG fractions of antisera raised against Fc₅μ and Fcμ′ fragments. cμ3 and cμ4 fragments had been prepared from human IgMк (Key) by hot trypsin digestion. Haemagglutination inhibition tests showed that the cμ4-binding fraction only reacted with cμ4 fragments. The cμ3-binding fraction reacted with cμ3 fragments but showed a minor reaction with cμ4 fragments. Immunization with Fcμ′ fragments predominantly yielded antibodies against the cμ3 domain, whereas immunization with Fc₅μ fragments yielded antibodies more directed against the cμ4 domain. Immunization with isolated cμ4 fragments led to the production of antibodies which reacted with the isolated cμ4 domain but not with the cμ4 domain within the larger structures of Fcμ′ or Fc₅μ fragments.     

Synthesis and characterization of dextran radiocontrast carriers, 1. Ether methylamide derivatives

The synthesis of dextran radiocontrast carriers was undertaken to obtain derivatives expected to have slow and controlled biodegradation as well as slow extravascular diffusion features. The results presented here particularly concern derivatives including a methylamide spacer, prepared by condensation of the amine function of the contrast product with carboxymethylated dextran (CMD). The effect of various coupling reagents on the final yields is disucssed. Concerning the macromolecular features, all the synthesized products were characterized by acid-base titration, elemental analysis, flame photometry, spectroscopic determinations and gel-permeation chromatography. GPC showed that the carboxymethylation of dextran leads to some reticulation (doubling the molecular weights), whereas the coupling of CMD with the contrast compound does not alter the molecular weight and the polydispersity index of the products appreciably.     

Synthesis of side-chain liquid-crystalline polyethers by chemical modification of polyepichlorohydrin

Copolymers derived from polyepichlorohydrin (PECH), bearing pendant mesogenic units are obtained by chemical modification of atactic samples of PECH with sodium 4-cyano-4′-biphenoxide. Rate and yield of substitution are strongly dependent on the molecular weight of the used PECH. A substantial rise (ΔT ～ 110°C) of glass transition temperature with the percentage of incorporated cyanobiphenyl groups is observed. Copolymers with an amount of substitution higher than 60% present thermotropic liquid-crystalline behaviour and form nematic phases. Chemical modification of PECH offers a simple method for the synthesis of new liquid-crystalline polyethers whose transition temperatures can be adjusted by varying the amount of substitution.     

Production and characterization of monoclonal antibodies specific for human mast cell tryptase

Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32.5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl DL-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.     

Derivation and characterization of a monoclonal hybridoma antibody specific for human alpha-fetoprotein

A monoclonal antibody specific for human alpha-fetoprotein (HAFP) was derived by the hybridoma technique. Spleen cells from mice immunized with pure HAFP were fused with a mouse myeloma cell line (P3×63-Ag-8) and an anti-HAFP secreting hybridoma cell line was cloned in soft agarose. The HAFP specific antibody was shown to be a monoclonal IgG₁ subclass with extremely high avidity for HAFP.     

Immunochemical characterization of binding sites of hybridoma antibodies specific for alpha (1 leads to 6) linked dextran

The combining sites of seven BALB/c IgM, four BALB/c IgA and one C57BL/6 IgA hybridoma antibodies specific for alpha (1 leads to 6) linked dextran were probed by precipitin and precipitin inhibition assays. The 12 antibodies are able to bind to linear determinants in the interior of the dextran molecule; some have sites complementary to six alpha (1 leads to 6) linked glucose residues and other have sites complementary to seven alpha (1 leads to 6) linked glucose residues. From the analysis of the precipitins and precipitin inhibitions, it is concluded that no two hybridoma proteins have identical binding sites.     

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12

Abstract: We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.     

Synthesis and characterization of pH-sensitive dextran hydrogels as a potential colon-specific drug delivery system.

pH-Sensitive dextran hydrogels were prepared by activation of dextran (T-70) with 4-nitrophenyl chloroformate, followed by conjugation of the activated dextran with 4-aminobutyric acid and cross-linking with 1,10-diaminodecane. The cross-linking efficiencies determined by mechanical measurements were in the range of 52-63%. Incorporation of carboxylpropyl groups in dextran hydrogels led to a higher equilibrium and faster swelling under high pH conditions. The swelling reversibility of hydrogels was also observed after repeated changes in buffers between pH 2.0 and 7.4. The slow rates of swelling and deswelling in response to changes in pH were attributed to the hydrophilic nature of dextran and formation of hydrogen bonds between the hydroxyl groups of dextran with water molecules. The pronounced effect of carboxylic acid content on degradation of hydrogels was observed after 4 h of incubation with dextranase and the influence significantly decreased after exposure to the enzyme for 8 h. The mechanism of bulk degradation of hydrogels under high swelling extent was substantiated using Coomassie blue protein assay. The release rate of bovine serum albumin from hydrogels was primarily determined by the swelling extent. The release rate was further enhanced by addition of dextranase in buffer solutions.     

Purification and characterization of human liver specific F antigen.

This paper reports the properties of purified human F antigen (liver-specific antigen). Homogenates of liver in 0-25 M sucrose were centrifuged at 105,000 g. The supernatants were chromatographed on Sepharose 6-B and four major peaks were separated. The third peak proved to be predominantly F antigen. This fraction was subsequently subjected to DEAE-cellulose column chromatography and F antigen was eluted at a concentration less than 0-2 M NaCl in 0-01 M sodium phosphate buffer (pH 7-2). Finally, purified F antigen was obtained after preparative isoelectric focusing. Purified human F antigen was found to have a mol. wt between 40,000 and 80,000, a pI of 6-5-6-7 and a density of 1-26. It is a protein antigen and contains no detectable carbohydrate or lipid. No differences were found in purified F-antigen preparations from several species when tested by sodium dodecyl sulphate (SDS) disc gel electrophoresis. Immunofluorescent studies showed that F antigen was homogeneously distributed in the cytoplasm of liver cells but was not present on the cell surface. Immunization of guinea-pigs with purified human liver-specific protein did not induce antibody to the F antigenic determinant defined by mouse anti-F antiserum. It did, however, induce antibodies to two human liver antigens. One of these seems to be a human-specific determinant on the F antigen molecule and the other appears to be a separate molecule which is similar in molecular weight and electrophoretic mobility to the F-antigen molecule.     

Production and characterization of monoclonal antibodies specific for a glycosylated polypeptide of human cytomegalovirus

Nine hybrid cell lines producing antibodies specific for cytomegalovirus (CMV) antigen were obtained after fusion of P3/X63-Ag8 myeloma cells with spleen cells from BALB/c mice immunized with CMV complement-fixing antigen. By the immunoblot technique, five of nine antibodies (4D11, 7B4, 7D2, 8E3, and 8E10) were identified as being reactive to a CMV glycosylated polypeptide with molecular weight of 66,000 (GP66). Four other antibodies (1B8, 8E9, 4D2, and 7E2) appeared to be reactive with CMV antigen(s) only if the antigen was not denatured by sodium dodecyl sulfate. These remain unassigned until further studies are done. With the enzyme-linked immunosorbent assay (ELISA), competitive bindings were performed with a constant amount of horseradish peroxidase-conjugated antibody and various concentrations of unconjugated homologous and heterologous antibodies on CMV antigen-coated ELISA wells, and the antigenic determinant specific for each antibody was determined. The nine antibodies could be classified into six different groups, each group reacting with a different epitope or a different region with two or more antigenic determinants which are so close to each other that they cause binding inhibition. They are groups A (4D11), B (7B4, 8E10), C (7D2), D (4D2, 7E2, 8E9), E (8E3), and F (1B8). The extent of competition among antibodies within each group was the same. By using the two antibodies that reacted with different epitopes on GP66, a double-antibody sandwich ELISA method was developed. The method was sensitive enough to detect as little as 50% of the antigen present in one infected cell or 0.000245 U of CMV complement-fixing antigen per test well. Other strains of CMV (David, Kerr, Espilat, C-87, and five clinical isolates) gave positive results, whereas herpes simplex virus types 1 and 2, varicella-zoster virus and Epstein-Barr virus nuclear antigen preparations did not. By the indirect immunofluorescence assay, antibodies 4D11 and 8E3 were able to detect GP66 in the nucleus of CMV-infected F-5000 human embryonic fibroblasts as early as 2 h postinfection and were superior in this respect to the remaining seven antibodies tested. By the double-antibody sandwich ELISA, the presence of GP66 in CMV-infected cells was detected as early as 2 h postinfection.     

The effects of chemical modification on the antigenicity of human and rabbit immunoglobulin G.

In order to characterize the precise structure within human and rabbit IgG molecules against which 'general' rheumatoid factors are directed, an immunochemical comparison has been made of the effects of the selective substitution of specific amino acid side-chains on various types of antigenicity exhibited by human and rabbit IgG. The epsilon-amino groups of lysine residues have been substituted by citraconylation and carbamylation; whilst tyrosine residues have been substituted by nitration with tetranitromethane. In this manner, evidence has been obtained which indicates that the autoantigenic determinants of human IgG are structurally distinct from species-specific ones and from certain Fc-located allotypic markers (Gm(a) and Gm(x)). It is also concluded that lysine residues are probably not involved in the site of IgG reactivity with 'general' rheumatoid factors, in contrast to tyrosine residues which appear to be implicated in the activity of human but not rabbit IgG.     

Production and characterization of specific monoclonal antibodies of the human thyroid stimulating hormone

Spleen cells from BALB/c mice immunized with human thyroid stimulating hormone (beta-subunit) were fused with mouse myeloma cells (P3/X63-Ag8) and five hybridomas secreting monoclonal antibodies (MAbs) were obtained. These hybridomas specifically recognize (hTSH) and do not cross-react with the other human glycoprotein hormones such as: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hcG). The MAbs were of the IgG1 subclass and ascitic fluid from these hybridomas was purified by affinity chromatography on Protein A-sepharose CL-4B column to isolate the IgG1 active fraction. The affinity constant of these MAbs ranged from 3.2 x 10(10) to 1.5 x 10(11) M(-1).     

Contributions of negatively charged chemical groups to the surface-dependent activation of human plasma by soluble dextran derivatives

Negatively charged surfaces are known to promote contact activation. The mechanism responsible for increasing affinity for surfaces is not yet quite understood, although the presence of negative charge densities is thought to be a prerequisite. With the availability of soluble dextran derivatives, varyingly substituted with charged methylcarboxylate, methylbenzylamide sulphonate and uncharged methylbenzylamide residues, we were able to discriminate between the contributions of these chemical moieties to contact activation, thus suggesting that the stimulating properties of synthetic negatively charged surfaces should also be described in terms of specific interactions instead of global negative charge density. This could be effected by quantifying the activating capacities as a function of the chemical group composition. A direct correlation linking activating capacities to anticoagulant properties has been observed.     

Synthesis,chemical and rheological characterization of new hyaluronic acid-based hydrogels

New hyaluronic acid-based hydrogels have been synthesized. The carboxylate groups of hyaluronan were activated in order to bind the amino terminal groups of the di-amine cross-linking reagent. Different hydrogels were obtained according to the different di-amine cross-linking agents (1,3-diaminepropane, 1,6-diaminohexane, PEG500 di-amine, and PEG800 di-amine). The crosslinked polymer (C.L.Hyal) was then sulphated (C.L.HyalS) by a heterogeneous reaction using sulphur trioxide pyridine complex (SO₃-Py). The thermo-mechanical properties and swelling degree were evaluated and are discussed in relation to the chemical structure and the hydrophilic character of the gels. The different behaviours of C.L.Hyal and C.L.HyalS indicate the important role of sulphated groups.