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Biomedical subjects

N E Nifant'ev

Publications and source records attributed to N E Nifant'ev.

At least 19 recordsLinked to original sources
PubMed2002

Human plasma trans-sialidase donor and acceptor specificity.

Earlier we have isolated from human plasma desialylated low density lipoproteins (dLDL) and showed that, first, dLDL induce cholesterol esters accumulation--the main process accompanying atherosclerosis development. Second, the process of lipoprotein desialylation took place in plasma, and, finally, sialic acids removed from LDL are transferred to other serum glycoconjugates. In this study we have isolated from human plasma an enzyme transferring sialic acid residues (trans-sialidase) by affinity chromatography and studied its donor and acceptor specificity. Isolated enzyme in the presence of saccharide-acceptor can remove sialic acids from different lipoproteins, glycoproteins (fetuin, transferrin), and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Plasma enzyme translocates alpha2-6, alpha2-3 and to a lower extent alpha2-8 bonded sialic acids. Sialoglycoconjugates of human serum erythrocytes, serum lipoproteins, glycoproteins, and gangliosides can serve as donors of sialic acid for trans-sialidase. Desialylated lipoproteins, especially dLDL, are more preferable sialic acid acceptors. Transferred sialic acid is found to be alpha2-6, alpha2-3, and alpha2-8 connected.

Humans↗
PubMed2001

Synthesis of Neu5Ac- and Neu5Gc-alpha-(2-->6')-lactosamine 3-aminopropyl glycosides.

In order to prepare 3-aminopropyl glycosides of Neu5Ac-alpha-(2-->6')-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-alpha-(2-->6')-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4',6'-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.

Carbohydrate Conformation↗
PubMed2000

Synthesis of 3-O-sulfoglucuronyl lacto-N-neotetraose 2-aminoethyl glycoside and biotinylated neoglycoconjugates thereof.

The 2-aminoethyl glycoside of pentasaccharide 3-O-sulfo-GlcA(beta-1-->3)Gal(beta-1-->4)GlcNAc(beta-1-->3)Gal(beta-1--> 4)Glc(beta (1) and its conjugates with biotin and biotinylated polyacrylic acid were synthesized as molecular probes to investigate the recognition of the HNK-1 epitope containing carbohydrates by proteins. Key steps in the first of two investigated schemes for the preparation of the target compound 1 were (a) assembling of the pentasaccharide backbone (compound 10) by glycosylation of selectively substituted allyl glycoside of the trisaccharide GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta with glucuronyl-galactose glycosyl donor, (b) transformation of the allyl aglycon in 10 into 2-azidoethyl one (to give 11), (c) selective deprotection of the OH group at C-3 of the GlcA residue in 11 via saponification, intramolecular formation of 6,3-lacton (13) and its methanolysis, and (d) subsequent O-sulfation. The alternative scheme with the use of 2-azido-ethyl glycoside of the trisaccharide GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta instead of the allyl glycoside 6 was less effective due to smaller yield at the step of pentasaccharide synthesis. Additionally to 1 the 2-aminoethyl glycosides of the oligosaccharides GlcA(beta-1-->3)Gal(beta-1-->4)GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta, 3-O-sulfo-GlcA(beta-1-->3)Gal(beta, and GlcA(beta-1-->3)Gal(beta were also synthesized.

Acrylic Resins↗
PubMed2000

Antitumour activity of cytotoxic liposomes equipped with selectin ligand SiaLe(X), in a mouse mammary adenocarcinoma model.

The overexpression of lectins by malignant cells compared with normal ones can be used for the targeting of drug-loaded liposomes to tumours with the help of specific carbohydrate ligands (vectors). Recently we have shown that liposomes bearing specific lipid-anchored glycoconjugates on a polymeric matrix bind in vitro to human malignant cells more effectively and, being loaded with a lipophilic prodrug of merphalan, reveal higher cytotoxic activity compared with unvectored liposomes. In this study, carbohydrate-equipped cytotoxic liposomes were tested in vivo in a mouse breast cancer model, BLRB-Rb (8.17)1Iem strain with a high incidence of spontaneous mammary adenocarcinoma (SMA). Firstly, a cell line of the SMA was established which was then used to determine the specificity of the tumour cell lectins. After screening of the lectin specificity of a number of fluorescent carbohydrate probes, SiaLe(X) was shown to be the ligand with the most affinity, and a lipophilic vector bearing this saccharide was synthesised. Then different liposomal formulations of the synthetic merphalan lipid derivative and SiaLe(X) vector were prepared and applied in the treatment of mice with grafted adenocarcinomas. The results of the tumorigenesis data show that the therapeutic efficacy of merphalan increases sharply after its insertion as a lipophilic prodrug into the membrane of SiaLe(X)-vectored liposomes.

Adenocarcinoma↗
PubMed2000

Xenotransplantation: in vitro analysis of synthetic alpha-galactosyl inhibitors of human anti-Galalpha1-->3Gal IgM and IgG antibodies.

Pig-to-human xenotransplantation might be an option to overcome the increasing shortage of human donor organs. However, naturally occurring antibodies in human blood against the Galalpha1-->3Gal antigen on pig endothelial cells lead to hyperacute or, if prevented, acute or delayed vascular rejection of the pig graft. The purpose of this study was therefore to evaluate synthetic oligosaccharides with terminal Galalpha1-->3Gal to inhibit antigen-binding and cytotoxicity of anti-alphaGal antibodies against pig cells. Different oligosaccharides were synthesized chemically and by a combined chemico-enzymatic approach. These included monomeric di-, tri-, and pentasaccharides, a polyacrylamide-conjugate (PAA-Bdi), as well as di-, tetra-, and octamers of Galalpha1-->3Gal. All were tested for inhibitory activity by anti-alphaGal ELISA and complement-dependent cytotoxicity tests. PAA-Bdi was the best inhibitor of binding as well as cytotoxicity of anti-alphaGal antibodies. Monomeric oligosaccharides efficiently prevented binding of anti-alphaGal IgG, but less well that of anti-alphaGal IgM, with tri- and pentasaccharides showing a better efficacy than the disaccharide. The two trisaccharides Galalpha1-->3Galbeta1-->4GlcNAc and Galalpha1-->3Galbeta1-->3GlcNAc were equally effective. Oligomers of Galalpha1-->3Gal were more effective than monomers in blocking the binding of anti-alphaGal IgG. However, they could not block IgM binding, nor could they match the efficacy of PAA-Bdi. We conclude that oligosaccharides with terminal Galalpha1-->3Gal, most effectively as PAA-conjugates, can prevent binding and cytotoxicity of human anti-alphaGal in vitro. The PAA-Bdi conjugate might be most suited for use as a Sepharose-bound immunoabsorption material.

Animals↗
PubMed2000

[Determination of substrate specificity of fucosyltransferase from rat brain using synthetic acceptors].

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Gal beta 1-4GlcNAc beta 1-R fragment were subjected to enzymic fucosylation. The isomer with a 1-3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated whereas Fuc alpha 1-2Gal beta 1-4GlcNAc displayed the same substrate properties as Gal beta 1-4GlcNAc. FT from cerebellum and forebrain was shown to have the specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from forebrain, the specificity profiles being similar.

Animals↗
PubMed1999

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]

Carbohydrate Conformation↗
PubMed1998

Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.

Acrylic Resins↗
PubMed1998

Conformational changes due to vicinal glycosylation: the branched alpha-L-Rhap(1-2)[beta-D-Galp(1-3)]-beta-D-Glc1-OMe trisaccharide compared with its parent disaccharides.

Conformations of the alpha-L-Rhap(1-2)-beta-D-Glc1-OMe and beta-D-Galp(1-3)-beta-D-Glc1-OMe disaccharides and the branched title trisaccharide were examined in DMSO-d6 solution by 1H-nmr. The distance mapping procedure was based on rotating frame nuclear Overhauser effect (NOE) constraints involving C- and O-linked protons, and hydrogen-bond constraints manifested by the splitting of the OH nmr signals for partially deuteriated samples. An "isotopomer-selected NOE" method for the unequivocal identification of mutually hydrogen-bonded hydroxyl groups was suggested. The length of hydrogen bonds thus detected is considered the only one motionally nonaveraged nmr-derived constraint. Molecular mechanics and molecular dynamics methods were used to model the conformational properties of the studied oligosaccharides. Complex conformational search, relying on a regular phi, psi-grid based scanning of the conformational space of the selected glycosidic linkage, combined with simultaneous modeling of different allowed orientations of the pendant groups and the third, neighboring sugar residue, has been carried out. Energy minimizations were performed for each member of the phi, psi grid generated set of conformations. Conformational clustering has been done to group the minimized conformations into families with similar values of glycosidic torsion angles. Several stable syn and anti conformations were found for the 1-->2 and 1-->3 bonds in the studied disaccharides. Vicinal glycosylation affected strongly the occupancy of conformational states in both branches of the title trisaccharide. The preferred conformational family of the trisaccharide (with average phi, psi values of 38 degrees, 17 degrees for the 1-->2 and 48 degrees, 1 degree for the 1-->3 bond, respectively) was shown by nmr to be stabilized by intramolecular hydrogen bonding between the nonbonded Rha and Gal residues.

Carbohydrate Sequence↗
PubMed1998

[Synthesis of oligosaccharides related to HNK-1 antigen. 2. Synthesis of 3'''-O-(3-O-sulfo-beta-D-glucuronopyranosyl)-lacto-N-neotetrao se beta-propylglycoside].

A derivative of allyl 3"-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-lactoside with a free OH group at C-4GlcNAc was glycosylated with trichloroacetimidate of selectively protected GlcA(beta 1-->3)Gal alpha disaccharide in dichloromethane in the presence of trimethylsilyl triflate resulting in a pentasaccharide product with an 82% yield. This product was converted to monohydroxy derivative with a free OH group at C-3GlcA via the formation and the subsequent opening of the 6,3-lactone ring in the glucuronic acid residue. The 3"'-O-sulfation of the monohydroxy derivative, the removal of the protective groups, and the reduction of the allyl aglycon yielded the pentasaccharide propyl glycoside NaSO3-3GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-Opr comprising the oligosaccharide chain of the SGGL-1 glycolipid, which is recognized by HNK-1 antibodies. NaSO3-3GlcA(beta 1--> 3)Gal beta OAll, GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-OPr and GlcA(beta 1-->3)Gal beta OAll were synthesized in a similar way.

CD57 Antigens↗
PubMed1997

Fucoidan inhibits leukocyte recruitment in a model peritoneal inflammation in rat and blocks interaction of P-selectin with its carbohydrate ligand.

Neutrophil recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin-mediated endothelial adherence. The effect of fucoidan (natural sulfated polymer of L-fucose) on the selectin dependent PMN migration into rat peritoneum following the induction of inflammation by peptone injection was studied. Peritonitis was characterized by an increase in the total cell number (from 45.3 x 10(6) to 91.6 x 10(6)/rat), and by highly elevated PMN content (from 0.2% to 58%) in the rat peritoneal cavity 3 h after peptone injection. Intravenous administration of fucoidan was found to reduce, in a dose-dependent manner, neutrophil migration into peritoneum. Fucoidan in a dose as low as 0.8 mg per rat caused 96.8% reduction of neutrophil extravasation. The inhibitory effect of fucoidan was also dependent on the time intervals between the peptone and fucoidan injections. The maximal inhibitory effect of fucoidan was observed within the first 15 min after the induction of peritonitis and it was maintained at a level of 80% during 1.5 h. Administration of fucoidan 2.5 h after peptone injection had practically no effect on PMN extravasation. Since P-selectin is known to play a key role at the earlier stages of PMN extravasation, it was suggested that the inhibitory effect of fucoidan was mostly due to its interaction with P-selectin. The in vitro experiments demonstrated the high affinity of fucoidan for both isolated P-selectin and P-selectin in plasma membranes of activated platelets.

Animals↗
PubMed1997

[Synthesis of oligosaccharides related to the HNK-1 antigen. Synthesis of selectively protected allyl-3-o-methyl(beta-D- glucopyranosyl)uronate-beta-D-galactopyranoside].

The glycosylation of several mono- and dihydroxyl glycosyl acceptors based on allyl beta-D-galactopyranoside with completely acylated glucuronyl bromides under the Helferich reaction conditions was studied in order to develop a method for the preparative synthesis of selectively protected disaccharide beta-D-GlcA-(1-->3)-beta-D-Gal in a form that can be used for further preparation of corresponding glycosyl donors and spacerated derivatives. We found that 1,2-orthoesters were the major primary products of the reaction, and their further conversion into isomeric glycosides depended on pH and can be regulated by the type of molecular sieves used. When Acid Washed Molecular Sieves AW 300 were used, glycosides were predominantly synthesized. No selective formation of the (1-->3)-bound disaccharide was observed upon glycosylation of glycosyl acceptors with 2,3- and 3,4-diol groupings. This (1-->3)-bound disaccharide was most efficiently synthesized by glycosylation of allyl 4,6-O-benzylidene-2-O-benzoyl-beta-D-galactopyranoside with pivaloylated glucuronyl bromide. With acetylated or benzoylated glucuronyl bromides or with pivaloyl glucuronyl imidate, this galactoside can also be glycosylated but with a lower yield of the target (1-->3)-bound disaccharide and lower glycosylation regioselectivity.

Allyl Compounds↗
PubMed1996

An E-selectin binding assay based on a polyacrylamide-type glycoconjugate.

Here we show that biotinylated polyacrylamide-type glycoconjugates which contain sialyl Lewis X (sLex-polymer) or sialyl Lewis A (sLea-polymer) are ligands for E-selectin. sLea-polymer bound E-selectin with higher affinity than sLex-polymer. Based on this property we used the sLea-polymer to establish a sensitive cell-free binding assay for the characterization of E-selectin antagonists. The assay involves complexation of the biotinylated sLea-polymer with streptavidin-peroxidase. This complex is incubated with E-selectin mouse Ckappa fusion protein immobilized onto microtiter plates. Bound complex is detected by the peroxidase reaction. sLea-polymer bound in a Ca2+-dependent manner consistent with the function of E-selectin as a C-type lectin. Control glycoconjugates with sialic acid (alpha-Neu5Ac), Lewis A (Lea), or beta-D-glucose residues instead of sLea failed to interact with the E-selectin. Neutralizing anti-E-selectin antibodies blocked completely binding to E-selectin. This demonstrates specificity of the assay system. sLex blocked binding of the sLea-polymer to E-selectin by 50% at a concentration of 550 microM (IC50). The assay was used to characterize sLea-polymers with differing sLea content as multivalent inhibitors of E-selectin binding. The inhibitory activity of these polymeric forms of sLea increased with their sLea content up to IC50s in the low micromolar range. The binding assay described is sensitive, rapid, and simple and of low variability. Therefore it should be advantageous for the identification and characterization of novel E-selectin antagonists.

Acrylic Resins↗
PubMed1996

Crystal and molecular structure of a histo-blood group antigen involved in cell adhesion: the Lewis x trisaccharide.

This work describes the first crystal structure ever reported of a histo-blood group carbohydrate antigen: Le(x). This study provides a detailed description of the conformation of two crystallographic independent molecules in a highly hydrated environment along with their hydrogen bonding properties and packing features. Some interactions observed between adjacent trisaccharides can provide the basis for involvement of Le(x)-Le(x) interactions in cell-cell adhesion.

Carbohydrate Conformation↗
PubMed1996

[Selectin receptors. 5. Monoclonal antibodies to the synthetic antigens SiaLe(a) and SiaLe(x)].

Five IgM class monoclonal antibodies (MAb) to the SiaLea tetrasaccharide, which is known as a serologic tumor marker CA 19.9, and three MAbs (one of IgG3 and two of IgM class) to the SiaLex tetrasaccharide (differentiation antigen CD15s) were obtained against totally synthetic immunogens. The epitope specificity of the antibodies was determined using a wide range of oligosaccharides and their polyacrylamide conjugates. MAb 4E10 against SiaLea and MAb 4G5 to SiaLex were highly specific to the antigen predefined by immunization: they did not cross-react either with structurally and conformationally related oligosaccharides or with their disaccharide fragments. Two MAbs to SiaLea (D7 and E5B1) showed a weak binding to SiaLex. MAb CC1 recognized SiaLea and SiaLex almost equally, and MAb 5H9 to SiaLea cross-reacted with the non-sialylated form, the Lea trisaccharide. Two MAbs against SiaLex A3 and B11 bound to all carbohydrate structures containing the core disaccharide Gal beta 1-3(4)GlcNAc.

Animals↗
PubMed1995

Human influenza virus recognition of sialyloligosaccharides.

Sialic acids are essential components of cell-surface receptors utilized by influenza viruses. To evaluate the recognition of asialic sugar parts of the receptor, three representative strains of human influenza A and B viruses were tested for their binding of a panel of sialyloligosaccharides. The highest affinity binding carbohydrate determinants recognized by the viruses in a context of different core structures were Neu5Ac alpha 2-3Gal for the type B virus, Neu5Ac alpha 2-6 Gal for the H3 subtype virus, and Neu5Ac alpha 2-6Gal beta 1-4GlcNAc for the H1 subtype virus. Penultimate to these determinants parts of the sialyloligosaccharides studied either contributed less significantly to the binding affinity, or interfered with the binding.

Binding Sites↗
PubMed1994

[Selectin receptors. 1. Synthesis of SiaLe(a) and SiaLe(x) tetrasaccharides and their polymeric conjugates].

Spacer-armed tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Fuc alpha 1- 4)GlcNAc-sp (SiaLe(a), sp = OCH2CH2CH2NH2) was synthesized from 6-BnGlcNAc-sp with 6-BnAc3GalSEt, Bn3FucSEt, and Ac4Neu5AcSEt methyl ester as glycosyl donors. The final glycosylation of the trisaccharide bearing unprotected 2-, 3-, and 4-hydroxyls of galactose moiety was promoted with NIS-TfOH in MeCN, yield of alpha-sialoside being 48% (29% as the NGlcNAc-SEt derivative). A mixture of beta-connected (2-3) and (2-4) lactones was obtained as a side product (30%). Tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-sp (SiaLe(x)) was synthesized by a similar way starting from 6-Bn-3-MeOBnGlcNAc-sp; yield of alpha-sialylation was 74%, neither N-SEt nor beta-connected lactones were isolated. Condensation of the spacered tetrasaccharides with poly(4-nitrophenylacrylate) gave N-substituted polyacrylamide-type polymers. Biotinylated probes and polymers modified with phosphatidylethanolamine (strong immunogens) were also obtained. The unsubstituted and labelled polymeric derivatives were used for studying selectins and other lectins, as well as production and epitope characterization of monoclonal antibodies against sialooligosaccharides.

Carbohydrate Sequence↗