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Biomedical subjects

R Brossmer

Publications and source records attributed to R Brossmer.

At least 37 records · Page 2Linked to original sources
PubMed1995

Suppression of influenza virus infection by an N-thioacetylneuraminic acid acrylamide copolymer resistant to neuraminidase.

We have previously shown that alpha-2-O-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2Me) has a higher affinity to bromelain-treated hemagglutinin (HA) of influenza A virus than sialic acid from natural sources (Machytka et al., 1993, FEBS Lett. 334, 117-120). We have now compared the inhibitory effects of alpha-Neu5thioAc2Me and other sialic acid analogs on receptor binding and plaque formation of intact influenza A viruses. When alpha-Neu5thioAc2Me was polymerized by conjugation to polyacrylamide, its affinity to HA increased 10(3)-fold. When analyzed by plaque reduction, the alpha-Neu5thioAc2 polymer was about 10 times more efficient as an inhibitor of virus replication than the alpha-Neu5Ac2 polymer, stressing the importance of sulfur at C5. The S-glycoside alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) had the same affinity to HA as alpha-Neu5thioAc2Me, but was resistant to neuraminidase. The alpha-Neu5thioAc2S polymer interfered with the replication of a wider spectrum of influenza A virus subtypes than the alpha-Neu5thioAc2 polymer. The results indicate that the alpha-Neu5thioAc2S polymer has the potential to be used as an inhibitor of influenza virus infection.

Acrylic Resins↗
PubMed1995

Enzymatic transfer of sialic acids modified at C-5 employing four different sialyltransferases.

We present kinetic studies on the enzymatic transfer of several synthetic sialic acid analogues, modified at C-5, to distinct glycoprotein glycans by sialyltransferases differing in acceptor- and linkage-specificity. Biochemical properties of sialic acids were modified by introducing formyl-, trifluoroacetyl-, benzyloxy-carbonyl-, and aminoacetyl-groups to the amino group at C-5 of neuraminic acid. The latter substitution renders the corresponding alpha-glycoside resistant towards sialidases. The respective CMP-sialic acid analogues were prepared by CMP-sialic acid synthase with a yield of 13-55%. The kinetic parameters of several sialyltransferases for the 5-substituted CMP-glycosides differed significantly. Relative to parent CMP-NeuAc, reaction rates of human- and rat liver Gal beta 1, 4GlcNAc alpha 2,6-sialyltransferases ranged from 50 to 170%, of GalNAc alpha 2,6-sialyltransferases from 40-140%, and of Gal beta 1,3Gal-NAc alpha 2,3-sialyltransferase from 20-50%. Resialylation of asialo-alpha 1-acid glycoprotein by 5-N-formyl- and 5-N-aminoacetyl-neuraminic acid employing rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase proceeded to about 80% of galactose sites which is identical to the extent achieved with parent NeuAc. According to our data, neosialoglycoconjugates which carry sialic acids modified at the N-acetyl group can be prepared for structure-function analysis, as this position seems crucial for recognition of adhesion proteins and influenza viruses.

Carbohydrate Conformation↗
PubMed1995

Sialic acid-binding lectins: submolecular specificity and interaction with sialoglycoproteins and tumour cells.

We examined the specificity of limulin, Limax flavus agglutinin (LFA) and Sambucus nigra agglutinin I (SNA I) at the submolecular level of sialic acid, and characterized their interactions with a panel of structurally distinct sialoglycoproteins. In haemagglutination inhibition assays NeuAc-alpha-glycosides were stronger inhibitors for limulin and LFA than native N-acetylneuraminic acid (NeuAc). The N-acetyl of NeuAc was crucial for binding to both lectins. N-thioacetylated NeuAc lost affinity for LFA, but still bound to limulin. Thus, distinct intermolecular interactions are involved in binding of sialic acid to the lectins. The glyceryl side chain was required for interaction with LFA, but not with limulin. SNA I specifically bound NeuAc alpha 2 --> 6Gal beta 1 --> 4Glc, but not monomeric sialic acids. Limulin and LFA strongly interacted with O-chain glycoproteins, whereas SNA I preferred N-chain proteins that carry NeuAc alpha 2 --> 6 residues. The lectins were compared with those from Cepaea hortensis and Tachypleus tridentatus (TTA) and to wheat-germ agglutinin, and were then used to probe tumour cell lines for cell surface sialylation. With the exception of TTA, all lectins interacted with the tumour cells. Limulin distinguished between the low (Eb) and highly (ESb) metastatic mouse lymphoma lines by selectively agglutinating sialidase-treated ESb cells.

Animals↗
PubMed1994

Site-specific labelling of the oligosaccharide chains of antibodies.

This paper presents a new method for site-specific labelling of antibodies employing enzymatic reactions without oxidizing or reducing agents. IgG was first treated with immobilized sialidase from Clostridium perfringens to cleave bound NeuAc. CMP-9-deoxy-9-salizoyl-NeuAc, an activated sialic acid analogue, was labelled with 131I via the iodogen-method in high yields (> 95%). Then the oligosaccharide chains of antibodies were labelled yield with the radioactive NeuAc analogue by transfer using alpha-2,6-sialyltransferase from rat liver in 50%.

Animals↗
PubMed1994

A lectin from the Asian horseshoe crab Tachypleus tridentatus: purification, specificity and interaction with tumour cells.

A lectin from the haemolymph of the Asian horseshoe crab Tachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-bound N-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues, N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin the N-acetyl group and the glyceryl side chain of N-acetylneuraminic acid are important, while presence of an aglycon, specially an alpha-glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin and Collocalia mucin, all being O-chain glycoproteins but carrying completely different carbohydrate chains. The majority of N-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the 'Tachypleus tridentatus agglutinin-receptor' which is present on red cells and O-chain glycoproteins.

Amino Sugars↗
PubMed1994

The differences in significance of alpha 2,3Gal-linked and alpha 2,6GalNAc-linked sialic acid residues in blood group M- and N-related epitopes recognized by various monoclonal antibodies.

The blood group M and N determinants of glycophorin A (GPA) contain O-linked oligosaccharide chains with alpha 2,3Gal-linked and alpha 2,6GalNAc-linked sialic acid residues which are required for the activity of most epitopes recognized by various anti-M and anti-N antibodies. In order to check whether these two types of sialic acid residues differ in their contribution to antigenic properties, the GPA-M and GPA-N preparations with monosialylated oligosaccharide chains were obtained and tested for binding of anti-M and anti-N monoclonal antibodies (MAbs). The GPAs with sialic acid residues linked to Gal (GPA2,3) were obtained by selective resialylation of asialoGPAs with alpha 2,3-sialyl-transferase. These preparations were tested by inhibition of binding of MAbs to enzyme-linked immunosorbent assay (ELISA) plates coated with the respective untreated target antigens. The GPAs with sialic acid residues linked to GalNAc (GPA2,6) were generated by treating GPAs adsorbed on ELISA plates with Newcastle disease virus (NDV) isolate (expressing sialidase specific for alpha 2,3Gal linkage), which was followed by testing the binding of MAbs to NDV-treated antigens. Different patterns of activity were obtained among 14 MAbs specific for sialic acid-dependent epitopes (eight anti-M and six anti-N). The results indicated that at least half of the MAbs showed distinct requirements for the presence of only one of two kinds of sialic acid residues (Gal or GalNAc linked) in the epitope. Only four MAbs (two anti-M and two anti-N) did not react with any of the 'monosialylated' forms of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylgalactosamine↗
PubMed1994

A new method for site-specific labelling of the oligosaccharide chain of antibodies: preliminary results.

There are various approaches for improving endoradiotherapy and diagnosis with monoclonal antibodies in nuclear medicine. The known methods of site-specific labelling of biomolecules based either on reactions with sulfhydryl groups or on reactions with aldehyde groups of the oligosaccharide chains effect unwanted alterations of the biomolecules. We present a new method to introduce radioactive halogens into the oligosaccharide chains of an antibody, based on the enzymatic transfer of the labelled synthetic sialic acid derivative CMP-9-deoxy-9-salizoyl-NeuAc. It was first labelled by the iodogen-method (iodine) in yields of more than 90%. Under selected conditions it was possible to obtain di- and trihalogenated products. Then the radioactive sialic acid derivatives were transferred during 90 minutes at room temperature with 2.6-sialyltransferase from rat liver into the oligosaccharide chains of antibodies. It is necessary to use neuraminidase pretreated antibodies with an increased number of binding sites for sialic acid derivatives. Yields of about 55% were obtained for the monoiodinated sialic acid derivative. With this method we present a reasonable alternative reaction of labelled compounds with biomolecules. Studies of the immunoreactivity are now in progress.

Animals↗
PubMed1993

Methyl alpha-glycoside of N-thioacetyl-D-neuraminic acid: a potential inhibitor of influenza A virus. A 1H NMR study.

The binding of influenza A virus hemagglutinin to its cell surface receptor, alpha-linked 5-N-acetylneuraminic acid (sialic acid), was studied in solution. The effect of structural modifications introduced into the N-acetyl group of the sialic acid on the binding was monitored by determining the dissociation constants by proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl alpha-glycoside of N-thioacetylneuraminic acid showed high, whereas the corresponding N-methylcarbamoylneuraminic acid exhibited relatively low binding affinity towards the hemagglutinin.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

A sialic acid analogue acting as a receptor determinant for binding but not for infection by influenza C virus.

We describe a synthetic sialic acid analogue, 9-thioacetamido-N-acetylneuraminic acid (9-thioacetamido-Neu5Ac), which is recognized by the receptor-binding activity of influenza C virus, but is resistant to the receptor-destroying enzyme (acetylesterase) of this virus. Following transfer of the analogue to the surface of receptor-negative cells, influenza C virus is able to attach to these cells, but is unable to infect the cells. This result suggests that inactivation of virus receptors by the receptor-destroying enzyme is essential for initiation of infection. Because of their unique properties such analogues promise to be powerful chemotherapeutic agents.

Acetylesterase↗
PubMed1993

Modification of cell surfaces by enzymatic introduction of special sialic acid analogues.

Taking advantage of the defined acceptor specificity of rat liver alpha 2,6-sialyltransferase, N-linked Gal beta 1,4GlcNAc-sequences of glycoconjugates at the plasma membrane of viable human erythrocytes and lymphoblastic IM-9 cells were modified to terminate in three NeuAc analogues endowed with special biochemical properties: sialidase-resistant 9-amino-NeuAc, O-acetylesterase-resistant 9-acetamido-NeuAc and photolabile 9-azido-NeuAc. Incorporation of the latter analogues and of parent NeuAc proceeded to similar values, whereas 9-amino-NeuAc yielded slightly reduced values.

Cells, Cultured↗
PubMed1992

Structure of the N- and O-glycans of the A-chain of human plasma alpha 2HS-glycoprotein as deduced from the chemical compositions of the derivatives prepared by stepwise degradation with exoglycosidases.

The structure of the glycans of the A-chain of human plasma alpha 2HS-glycoprotein was established from the chemical compositions of its derivatives prepared by sequential enzymatic degradation of the carbohydrate moiety, from the determination of the kind and amount of the monosaccharides liberated after each step of the enzymatic digestion, and from the distinct specificity of the highly purified exoglycosidases. The exoglycosidases were three sialidases (Vibrio cholerae, fowl plague virus, and Arthrobacter ureafaciens), two beta-galactosidases (Streptococcus pneumoniae and bovine testis), one alpha-N-acetylgalactosaminidase, one beta-N-acetylglucosaminidase, and one alpha-mannosidase. Utilizing sialidases with different cleavage specificities, the number of alpha 2-3- and alpha 2-6-linked sialic acid residues could be separately determined. As to the beta-galactosidases, the enzyme isolated from S. pneumoniae cleaves only beta 1-4-linked galactose residues, whereas the bovine testes enzyme acts on both the beta 1-4- and beta 1-3-linked galactose residues. Jack bean beta-N-acetylglucosaminidase cleaves beta 1-2, beta 1-4, and beta 1-6 GlcNAc with higher activity for the beta 1-2. Jack bean alpha-mannosidase cleaves alpha 1-2, alpha 1-6, and alpha 1-3 Man with greater activity for alpha 1-2 and alpha 1-6. Bovine liver alpha-N-acetylgalactosaminidase cleaves O-linked GalNAc. On the basis of these results, the A-chain of alpha 2 HS-glycoprotein was found to possess two biantennary N-glycans and two O-linked trisaccharides.

Blood Proteins↗
PubMed1992

A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.

Acetylesterase↗
PubMed1992

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.

Animals↗
PubMed1992

Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.

Animals↗
PubMed1992

Use of sialic acid analogues to define functional groups involved in binding to the influenza virus hemagglutinin.

The initial step of influenza infection is binding of the virus particles via their hemagglutinin to cell-surface sialic acids. This study was initiated to elucidate the functional groups of the nine-carbon sialic acid molecule which interact with the hemagglutinin and contribute to the affinity of this sugar to the protein. In order to address this question, synthetic sialic acid analogues were tested in a virus adsorption inhibition assay for their inhibitory potency. Modifications in three regions of the sialic acid molecule were evaluated: the glycerol side chain (C7-C9), the N-acetyl group at C5, and the carboxy group (C1). In the glycerol side chain, the hydroxy groups at C7 and C8 appear to be important for binding through hydrogen bonds, whereas the hydroxyl at C9 does not appear to be involved. The N-acetyl group is critical for the interaction of sialic acid with the hemagglutinin. The results suggest that its contribution is mediated through hydrophobic interactions of the methyl group. Finally, the orientation of the carboxy group is essential for the binding of sialic acid to the hemagglutinin. The information gained in this study will be useful in developing novel compounds which bind more avidly to the influenza virus hemagglutinin. Such a strategy may contribute to the design of new anti-influenza drugs.

Binding Sites↗
PubMed1992

Separation of tumor cells from a suspension of dissociated human colorectal carcinoma tissue by means of monoclonal antibody-coated magnetic beads.

Dissociation of colorectal tumor tissue directly obtained at surgery yields a cell suspension which contains tumor cells, leukocytes and erythrocytes. Coupling of a murine monoclonal antibody, HEA125, specific for human epithelial cells, to magnetic beads permits positive selection of a population containing essentially only tumor cells. Removal of cells from the beads is possible by papain treatment. Negative selection of tumor cells by means of beads coated with a leukocyte binding antibody offers no advantage since dead cells and erythrocytes remain.

Adenocarcinoma↗