[Preparation of biologically transformed raw material from woolly foxglove Digitalis lanata Ehrh and isolation of digoxin from it]. / Poluchenie biotransformirovannogo syr'ia naperstianki sherstistoi Digitalis lanata Ehrh i vydelenie iz nego digoksina.

A biotechnological approach is proposed for conservation of a terraneous part of woolly foxglove under anaerobic conditions with a subsequent air-sun drying of the biologically transformed raw material. During the conservation primary foxglove glycosides completely convert to secondary ones which do not transform further. A simple method is described for preparation from the transformed raw material of an enriched glycoside fraction with the yield of 3.6% and for isolation from this fraction of highly purified digoxin with the yield of 0.06% of the starting raw material, and the other secondary glycosides can be also isolated.

[Glycosylation of glycoconjugates on thymocyte surfaces]. / Glikozilirovanie glikokon''iugatov poverkhnosti timotsitov.

The ectosialation and ectogalactosylation of mouse thymocyte surface were studied. The incorporation of labeled monosaccharides (N-acetylneuraminic acid and galactose) into cell surface glycoproteins and glycolipids were demonstrated. Identification of glycolipids was carried out. The effect of glycosylation on the immune properties of thymocytes was established.

[Fluorescent derivatives of carbohydrates in the analysis of the structure of glycoconjugates. N-(4-methylcoumarin-7-yl) glycamines in the study of asparagine-bound carbohydrate chains of glycoproteins]. / Fluorestsentnye proizvodnye uglevodov v strukturnom analize glikokon''iugatov. N-(4-metilkumarin-7-il)glikaminy v izuchenii asparaginsviazannykh uglevodnykh tsepei glikoproteinov.

4-(N-Methylcoumarin-7-yl) glycamines were employed in studying asparagine-linked carbohydrate chains of acid desialylated fetuin. The procedure was optimised for the reductive amination of oligosaccharides with 7-amino-4-methylcoumarin in the presence of Na(CN)BH3 to lead to oligosaccharide glycamines (AMC-OS). AMC-OS were obtained from dextran oligosaccharides and from oligosaccharides released by hydrazinolysis of asparagine-linked sugar chains of asialofetuin. Reverse-phase HPLC and exclusion HPLC with fluorimetric quantitation of AMC-OS is described. TSK Gel 2000 SW column was calibrated using dextran AMC-OS to give linear relationship ln Ni = k(ti/tr)+b, where ti/tr is retention time of the AMC-OS relatively to the reference AMC-trisaccharide, and Ni is calibration unit value, characterizing molecular size of AMC-OS. Three AMC-OS, Gal3GlcNAc3Man3GlcNAc2-AMC (I) and (II), and Gal2GlcNAc3Man3GlcNAc2AMC (III), were obtained from asialofetuin in a molar ration of 1:1.8:0.1. Acid treatment of AMK-OS (II) in desialylation conditions also gave AMC-OS (III), thus suggesting a partial degalactosylation of the glycoprotein sugar chains during the desialylation. Consequent digestion of AMC-OS (II) and (III) with Jack bean beta-galactosidase and beta-N-acetylhexosaminidase led to the same AMC-OS, Man3GlcNAc2AMC. The final digestion product of AMC-OS (I) was GalGlcNAcMan3GlcNAc2AMC, suggesting a structural difference in one of the antennas of the minor sugar chain of asialofetuin. The monosaccharide quantitation and exoglycosidase sequencing were carried out at a 100 pmole level.

[Isomerization of trifluoroacetamidopropyl-2-acetamido-2-deoxy-D- galactopyranoside into alpha-anomer. A convenient method of synthesis of the artificial T-antigen]. / Izomerizatsiia triftoratsetamidopropil-2-atsetamido-2-dezoksi-beta- D-galaktopiranozida v alpha-anomer. Udobnyi metod sinteza iskusstvennogo T-antigena.

Bioside Gal beta 1-3GalNAc alpha 1-O(CH2)3NHCOCF3 has been synthesized. The key alpha-glycoside GalNAc alpha 1-O(CH2)3NHCOCF3 (peracetate) was obtained either by isomerization of its beta-anomer with trifluoromethanesulfonic acid, or by direct glycosylation of 3-(trifluoroacetamido)propanol with D-galactosamine (anomeric pentaacetate) in the presence of a mixture of trifluoromethanesulfonic acid and boron trifluoride etherate. De-O-acetylated alpha-galactosaminide obtained was further transformed into benzylidene derivative, the latter was glycosylated with acetobromogalactose to give the protected alpha-bioside. The removal of the protecting groups gave the (3-aminopropyl)-alpha-bioside, which was subsequently immobilized on bovine serum albumin and cytochrome c.

[Artificial peptide and carbohydrate antigens. Immobilization of haptens and adjuvant (MDP) on polyacrylamide]. / Iskusstvennye peptidnye i uglevodnye antigeny. Immobilizatsiia gaptenov i ad''iuvanta (MDP) na poliakrilamide.

To study the influence of the polyacrylamide carrier on immunogenic properties of the peptide and oligosaccharide haptens, we have prepared artificial antigens by conjugation of a synthetic hexapeptide (homologous to the fragment 95-100 of the murine H-2Db antigen heavy chain) or of an oligosaccharide (antigenic determinant of human blood groops, Lea) with polyacrylamide. In some cases the conjugates containing also a synthetic glycopeptide adjuvant, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), were used. Antisera against haptens were obtained by immunization of BALB/c mice with corresponding conjugates. By the enzyme-linked immunosorbent assay it was shown that these antisera had a high binding titer (up to 10 000) to corresponding hapten, and MDP immobilized on the same carrier as hapten possessed a considerable immunostimulating activity. Thus, usefulness of polyacrylamide for preparation of immunogenic artificial molecules carrying peptide and oligosaccharide haptens was demonstrated.

[Monospecific antibodies against synthetic T-antigen. Characteristics of their specificity and use in the identification of T-antigenic determinants on the cell surface]. / Monospetsificheskie antitela k iskusstvennomu T-antigenu. Kharakteristika ikh spetsifichnosti i primenenie dlia identifikatsii T-antigennykh determinant na poverkhnosti kletok.

Monospecific antibodies directed to a Thomsen-Friedenreich antigen (T-antigen) were obtained using artificial antigen. T-antigen immunodominant alpha-disaccharide Galbeta (1----3) GalNAc alpha 1-(T alpha) and its beta-anomer Gal beta (1----3) GalNAc beta 1-(T beta) were bound to bovine serum albumin (BSA) and cytochrome C (CCC) through a spacer (sp = -O(CH2)3NHCO (CH2)4CO-) by the azide method to give neoglycoproteins T alpha-sp-BSA, T alpha-sp-CCC and T beta-sp-BSA. Anti-T alpha antiserum was obtained by immunization of rabbits with T alpha-sp-BSA and then purified by sequential affinity chromatography on BSA-Sepharose and T alpha-sp-BSA-Sepharose to yield monospecific anti-T IgG antibodies. As elucidated by ELISA method, binding T alpha-sp-BSA to the antibodies was inhibited by T alpha-sp-CCC, T alpha-sp-OEt, asialofetuin, T alpha-OBzl, the activity of the inhibitors decreasing in the above order. Methyl beta-galactopyranoside, benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside, disaccharide Gal beta (1----3) GalNAc and H-sp-BSA were inactive. The inhibitory analysis suggests that both disaccharide moiety T alpha- and a definite part of the spacer are important for the binding and that T alpha-OCH2 seems to be the minimal recognized structure. In immunoprecipitation tests the antibodies react with T alpha-sp-BSA but not with T beta-sp-BSA, whereas peanut (Arachis hypogaea) lectin (PNL) precipitated both T alpha- and T beta-sp-BSA. These data suggest the significance of the alpha-galactosaminide bond for the antibody recognition. Desialylated human erithrocites (natural T-antigen) were effectively agglutinated with the antibodies. Murine cortical thymocytes (obtained by agglutination-sedimentation method using PNL) were agglutinated with the antibodies only partially (67%), while these cells as well as the cells unaffected by the antibodies were completely agglutinated with PNL. These results indicate to different contents of glycoproteins (T alpha) and glycolipids (T beta) oligosaccharide determinants on the surface of cortical thymocytes species.

[A convenient synthon for the synthesis of the determinant oligosaccharides Le and ABH (type 1)]. / Udobnyi sinton dlia polucheniia determinantnykh oligosakharidov Le i ABH (tip 1).

Precursors of blood group oligosaccharides ABH (type 1) and Le have been synthesized starting from benzyl 2-acetamido-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-6-O-ben zyl-2- deoxy-alpha-D-glucopyranoside after simple blocking and deblocking steps. The above disaccharide was prepared by regioselective galactosylation of benzyl 2-acetamido-6-O-benzyl-2-deoxy-alpha-D-glucopyranoside under Helferich conditions in 69% yield.

[Synthesis of determinant oligosaccharides of the ABH (type 1) blood group antigen and Leb tetrasaccharide from a common precursor]. / Sintez determinantnykh oligosakharidov gruppospetsificheskikh antigenov krovi sistemy ABH (tip 1) i tetrasakharida Leb iz obshchego predshestvennika.

Synthesis of blood group ABH (type 1) determinant oligosaccharides and Leb tetrasaccharide has been performed using the same trisaccharide precursor-benzyl 2-acetamido-4,6-O-benzylidene-[4,6-O-benzylidene-2-O-[2-O-benzyl-3,4-di- O- (4-nitrobenzoyl)-alpha-L-fucopyranosyl]-beta-D-galactopyranosyl]-2-deoxy - alpha-D-glucopyranoside. A- and B-determinants were prepared by alpha-galactosaminylation and alpha-galactosylation of the title trisaccharide, respectively. Leb-determinant was synthesized by a series of simple blocking and deblocking steps followed by alpha-fucosylation.

[Glycoproteins of tumor and normal tissue of the human breast]. / Glikoproteiny opukholevoi i normal'noi tkanei molochnoi zhelezy cheloveka.

Antigenic preparations from human tumour (TMG) and normal mammary gland (NMG) tissues were isolated, using water extraction followed by precipitation of proteins with perchloric acid, which were further separated into fractions A, B and C by gel filtration on Sepharose 6B. Glycoprotein fractions B and C were further separated by IEC on DEAE cellulose to give fractions B1-BIV and C1-CIV. A set of glycoproteins (Mr = 13 000-122 000; pI approximately 3-7.5) were detected in TMC and NMG, the glycoproteins specific for TMG or NMG as well as common for both TMG and NMG being found. The major glycoproteins (Mr = 46 000), i. e. GP-1 from TMG not found in NMG and GP-2 and GP-3 from NMG not found in TMG were isolated and characterized both biochemically and serologically.

[Purification and properties of beta-galactosidase from Alternaria tenius]. / Ochistka i svoistva beta-galaktozidazy Alternaria tenuis.

beta-Galactosidase from Alternaria tenius was purified to homogeneity from the cultural fluid using acetone precipitation, ion-exchange chromatography on DEAE-cellulose, adsorption on hydroxylapatite and affinity chromatography on N-(beta-D-galactopyranosyl-thiocarbamoyl)-beta-aminocaproyl-AN-Sepharose 4B. The enzyme homogeneity was demonstrated by ultracentrifugation and polyacrylamide gel electrophoresis with SDS or without it. The specific activity of the homogeneous enzyme is 160 u. per mg of protein; mol. weight as determined by various methods is 142 000-176 000, pI = 4.6, temperature optimum is 60-65 degrees, pH optima for o-nitrophenyl-beta-D-galactopyranoside (o-NPG) and lactose are 3.8--4.4 and 3.6--4.8, respectively. The Km values for o-NPG and lactose are 0.21 . 10(-3) and 6.57 . 10-3 M, respectively. The enzyme is a glycoprotein and contains up to 30% of carbohydrates. EDTA and pCMB have no effect on the beta-galactosidase activity. Galactose acts as a competitive inhibitor, while glucose has no inhibiting effect on the enzyme activity.

[Properties of membrane-bound sialyltransferase from rana temporaria liver]. / Izuchenie svoistv membranosviazannykh form sialiltransferaz iz pecheni liagushki Rana temproaria.

It was demonstrated that microsomal membranes from frog liver contain at least two different sialyltransferases involved in the synthesis of alpha 2 leads to 3 and alpha 2 leads to 6 oligosaccharide isomers. Studies on acceptor specificity of the sialyltransferase system with respect to low molecular acceptors revealed its similarity to the mammalian sialyltransferase system. However, sharp distinctions were observed in sialylation of mammalian glycoproteins. It was assumed that that the disaccharide unit of the acceptor oligosaccharide chain is a structural element, which in necessary but not sufficient for glycoprotein recognition by sialyltransferases.

[Lysosomal neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase of regenerating liver tissue]. / Izuchenie neiraminidazy, beta-galaktozidazy i beta-N-atsetilgliukozaminidazy lizosom regeneriruiushchei tkani pecheni

Neuraminidase (EC 3.2.1.18) beta-galactosidase (EC 3.2.1.23) and beta-N-acetylglucosaminidase (EC 3.2.1.30) were studied in normal and regenerating rat liver. All these glycosidases were shown to be predominantly localized in lysosome rich fraction. The activity of lysosomal neuraminidase in regenerating rat liver increased 24 hours after partial hepatectomy, whereas those of beta-galactosidase and beta-N-acetylglucosaminidase decreased. The presence of inhibitor of neurominidase in regenerating liver homogenate was shown. Actinomycin D and cycloheximide were shown to have no significant effect on lysosomal neuraminidase in regenerating rat liver.