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R Schauer

Publications and source records attributed to R Schauer.

At least 127 records · Page 7Linked to original sources
PubMed1992

The influence of alpha- and beta-galactose residues and sialic acid O-acetyl groups of rat erythrocytes on the interaction with peritoneal macrophages.

The significance of glycoconjugates on the surface of rat erythrocytes was studied in the interaction of these cells with homologous peritoneal macrophages. The erythrocytes exposing terminal alpha-galactose and thus of B blood group specificity, as well as sialic acid are not bound by the macrophages. beta-Galactose residues exposed by sialidase induced strong binding and additional alpha-galactosidase treatment enhanced the binding. beta-Galactose exposed on glycolipids after pronase and alpha-galactosidase treatment induced no binding. An intact protein core of the glycoproteins on the erythrocyte surface was necessary for interaction with macrophages. Partial de-O-acetylation of sialic acids prior to sialidase treatment stimulated subsequent binding of the erythrocytes.

Acetylation↗
PubMed1992

Interaction of rat peritoneal macrophages with homologous sialidase-treated thrombocytes in vitro: biochemical and morphological studies. Detection of N-(O-acetyl)glycoloylneuraminic acid.

Sialidase treatment of rat thrombocytes led to an increased binding of these cells to homologous peritoneal macrophages, but had no significant effect on the rate of phagocytosis during the experimental time. As revealed by electron microscopy, the partially desialylated thrombocytes adhere to macrophages predominantly via a small part of the membrane in a way that the discoidal cells adopt a vertical position with regard to the macrophage surface. One adherent macrophage was able to bind up to 55 sialidase-treated thrombocytes. Maximum binding was already reached after release of 13% of sialic acids. This interaction could be inhibited by free D-galactose and compounds with terminal D-galactose residues. Bound thrombocytes were released from the macrophages by treatment with lactose or EDTA. These experiments suggest that the interaction is mediated by a galactose-specific receptor on the macrophage surface and that galactose on thrombocytes is not recognized if it is masked by terminal sialic acid residues. The total sialic acid amount of the thrombocytes studied was about 70 micrograms sialic acid/10(10) cells being composed of 78% N-glycoloylneuraminic acid, 17% N-acetylneuraminic acid and 5% of the novel sialic acid N-(O-acetyl)glycoloylneuraminic acid, which was identified by mass spectrometry. Sixty-two percent of these sialic acids were susceptible to enzymic hydrolysis with Vibrio cholerae sialidase.

Animals↗
PubMed1991

2,3-Didehydro-2-deoxysialic acids structurally varied at C-5 and their behaviour towards the sialidase from Vibrio cholerae.

2,3-Didehydro-2-deoxy-N-trifluoroacetylneuraminic acid (5-trifluoroacetyl-Neu2en) (3) has been synthesised from Neu5Ac2en (1) by hydrazinolysis, to give Neu2en (2), followed by N-trifluoroacetylation. 2,3-Didehydro-2,3-dideoxy-D-glycero-D-galacto-2-nonulopyranoson ic acid (Kdn2en, 8) and 5-azido-2,3-didehydro-2,3,5-trideoxy-D-glycero-D-galacto-2-nonu lopyranosonic acid (5-azido-5-deoxy-Kdn2en, 9) have been prepared from the acetylated methyl esters of Kdn (4) and 5-azido-5-deoxy-Kdn (5) via Zemplén saponification. The behaviour of the above 2,3-didehydro-2-deoxysialic acids towards Vibrio cholerae sialidase has been investigated.

Kinetics↗
PubMed1991

Reversible binding of sialidase-treated rat lymphocytes by homologous peritoneal macrophages.

After sialidase treatment, lymphocytes disappear from the blood stream, but reappear after a few hours. The behavior of sialidase-treated rat lymphocytes was investigated by in vitro binding studies with homologous peritoneal macrophages. A lymphocyte mixture from thymus and spleen was treated with sialidase and cultured up to 55 h, and at various times, the binding of the lymphocytes to glass-adherent macrophages was studied by light and electron microscopy; vital lymphocytes were only bound but not phagocytosed, and the interaction with macrophages was inhibited by D-galactose. During culture of lymphocytes, either separately or with macrophages, the binding was more and more reduced, and a second sialidase treatment of cultured lymphocytes led again to increased binding which could be inhibited by D-galactose. This change did not occur in the presence of N-acetyl-2,3-didehydro-2-deoxyneuraminic acid, an inhibitor of sialidases, showing the sialic acid specificity of this phenomenon. Thus, the reversibility of lymphocyte binding could be explained by resynthesis of cell surface sialic acids.

Animals↗
PubMed1991

Application of sialidase antibodies for the diagnosis of clostridial infections.

Two immunological methods, a sialidase-inhibition test and an ELISA, had been developed for a species-specific detection of sialidases (EC 3.2.1.18) secreted by growing cells of Clostridium perfringens, C. septicum, and C. sordellii. These assays were applied to samples from the infected tissues and cotton wool plugs soaked with wound exudate from patients suspected to be suffering from gas gangrene. The results from 72 patients were compared with bacteriological investigations of 67 homologous samples. From these, 35 patients were found to be infected with one or two of the clostridial species: thirty patients contained C. perfringens, six samples showed an immunologically positive reaction for C. septicum and eight for C. sordellii. In the case of C. perfringens, a high degree of coincidence between the immunological and the bacteriological analyses was obtained. However, in infections with C. septicum or C. sordellii the bacteriological and immunological tests did not agree well. The simple and rapid sialidase inhibition test or the slightly more time-consuming ELISA, which even detects low quantities of sialidase, can be applied for the specific diagnosis of infections caused by C. perfringens, thus allowing a species-specific detection of this bacterial species in two or six hours, respectively, even at an early stage of infection. This early diagnosis is a prerequisite for a specific and life-saving therapy of this severe disease.

Bacterial Typing Techniques↗
PubMed1991

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins.

An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110,000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a sialidase from Clostridium chauvoei NC08596.

The sialidase secreted by Clostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10,200-fold, reaching a final specific activity of 24.4 U mg-1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn2+, Mg2+, and Ca2+ and bovine serum albumin have a positive effect on the sialidase activity, while Hg2+, Cu2+, and Zn2+, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.

Clostridium↗
PubMed1991

CMP-N-acetylneuraminic acid hydroxylase activity determines the wheat germ agglutinin-binding phenotype in two mutants of the lymphoma cell line MDAY-D2.

The dominant glycosylation mutants of MDAY-D2 mouse lymphoma cells, designated class 2 (D33W25 and D34W25) were selected for their resistance to the toxic effects of wheat germ agglutinin (WGA) and shown to express elevated levels of Neu5Gc. In accordance with this, the activity of CMP-Neu5Ac hydroxylase was found to be substantially higher in the mutant cells. The hydroxylase in the D33W25 mutant cells exhibited kinetic properties identical to those of the same enzyme from mouse liver. Growth rate experiments in vivo and in vitro, where the mutant cells grew more slowly at low cell densities in serum-free medium and also formed slower growing tumours in syngeneic mice, indicate that CMP-Neu5Ac hydroxylase expression may be associated with altered growth of the mutant cells.

Cell Division↗
PubMed1991

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland.

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio of N-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin. Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts of N-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content of N-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8 x 10(5) Da and aggregates in excess of 10(6) Da, while the minor mucin ranged from 3.0 x 10(5) to 10(6) Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.

Acetylation↗
PubMed1991

Panagrellus redivivus: failure to find evidence for the occurrence and biosynthesis of sialic acids.

The occurrence of sialic acids in the free-living nematode Panagrellus redivivus was studied by periodate oxidation/[3H]sodium borohydride reduction of about 10(7) nematodes. In parallel, the capability of sialic acid biosynthesis was examined by metabolic labeling of the same number of nematodes with N-[3H]acetylmannosamine. In both experiments, radioactivity was incorporated into the nematodes. Mild acid hydrolysis, however, did not release radioactively labeled sialic acids or derivatives as tested by radio thin-layer chromatography, suggesting that P. redivivus does not contain or synthesize sialic acids.

Animals↗
PubMed1991

Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages.

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater than or equal to 9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (Ki = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha 2----3 linkages when compared with alpha 2----6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha 2----3 linkages and to have a basic pI.

Amino Acids↗
PubMed1991

Cloning, sequencing and expression of the sialidase gene from Actinomyces viscosus DSM 43798.

Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac. The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A. viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa. The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species.

Actinomyces viscosus↗
PubMed1990

A study on the regulation of N-glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver.

The relative contribution of N-glycoloyl-beta-D-neuraminic acid (Neu5Gc) to total sialic acids expressed in mouse and rat liver glycoconjugates was found to be 95% and 11%, respectively. This considerable difference in sialic acid composition made these two tissues suitable models for a comparative investigation into the regulation of Neu5Gc biosynthesis and utilization. An examination of the CMP-glycoside specificity of Golgi-associated sialyltransferases using CMP-N-acetyl-beta-D-neuraminic acid (CMP-Neu5Ac) and CMP-Neu5Gc revealed no significant tissue-dependent differences. The Golgi membrane CMP-sialic acid transport system from rat liver did, however, exhibit a slightly higher internalisation rate for CMP-Neu5Ac, though no preferential affinity for this sugar nucleotide over CMP-Neu5Gc was observed. In experiments, where Golgi membrane preparations were incubated with an equimolar mixture of labelled CMP-Neu5Ac and CMP-Neu5Gc, no significant tissue-dependent differences in [14C]sialic acid composition were observed, either in the luminal soluble sialic acid fraction or in the precipitable sialic acid fraction, results which are consistent with the above observations. From this experiment, evidence was also obtained for the presence of a Golgi-lumen-associated CMP--sialic acid hydrolase which exhibited no apparent specificity for either CMP-Neu5Ac or CMP-Neu5Gc. The specific activity of the CMP-Neu5Ac hydroxylase, the enzyme responsible for the biosynthesis of Neu5Gc, was found to be 28-fold greater in high-speed supernatants of mouse liver than of rat liver. No hydroxylase activity was detected in the Golgi membrane preparations. It is therefore proposed that the cytoplasmic ratio of CMP-Neu5Ac and CMP-Neu5Gc produced by the hydroxylase, remains largely unmodified after CMP-glycoside uptake into the Golgi apparatus and transfer on to growing glycoconjugate glycan chains. The close relationship between the total sialic acid composition and the sialic acid pattern in the CMP-glycoside pools of the tissues lends considerable weight to this hypothesis.

Animals↗
PubMed1990

Antibodies to ADP-ATP carrier--an autoantigen in myocarditis and dilated cardiomyopathy--impair cardiac function.

The adenosine diphosphate (ADP)-adenosine triphosphate (ATP) carrier of the inner mitochondrial membrane is identified as an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain autoantibodies to the ADP-ATP carrier capable of inhibiting nucleotide transport in vitro. Recently, an antibody-related infringement of energy metabolism was shown in intact perfused hearts isolated from guinea pigs immunized with the ADP-ATP carrier. A decreased cytosolic-mitochondrial difference of the phosphorylation potential of ATP was measured that originated from a reduction in mitochondrial-cytosolic nucleotide transport. Nonimmunized animals did not show these changes in energy metabolism, despite being in a comparable metabolic state and performing equal external heart work. To establish whether antibodies to the ADP-ATP carrier can also alter cardiac function, hemodynamic parameters of isolated hearts of guinea pigs that were preimmunized with the carrier protein were measured. Cardiac metabolism was stimulated by exposing the hearts to a high calcium concentration in conjunction with a maximum elevation of the afterload. Mean aortic pressure, stroke volume, stroke work, and external heart work were found to be lowered significantly (p less than 0.005). The external heart work of the immunized hearts reached only about 20% of the level performed by control hearts. Myocardial oxygen consumption was lowered 2.5-fold, whereas the extent of lactate production was found to be more than doubled. These results show a diminished cardiac performance of hearts from animals immunized with the ADP-ATP carrier. Our findings demonstrate that autoimmunity to the ADP-ATP carrier may contribute to the pathophysiology of dilated cardiomyopathy as a subsequent stage of myocarditis by causing an autoantibody-mediated reduction in cardiac function on the basis of an imbalance between energy delivery and demand.

Animals↗
PubMed1990

Influence of sialic acids on the galactose-recognizing receptor of rat peritoneal macrophages.

The interaction of the galactose-recognizing receptor from rat peritoneal macrophages with ligands containing terminal galactose residues, such as asialoorosomucoid, desialylated erythrocytes or lymphocytes, can be inhibited by free N-acetylneuraminic acid (Neu5Ac) and oligosaccharides or glycoproteins containing this sugar in terminal position. This effect of Neu5Ac on the receptor is specific. The other naturally occurring or most of synthetic neuraminic acid derivatives tested do not exhibit an equivalent inhibitory potency as Neu5Ac. Although free Neu5Ac inhibits 5-fold stronger (K50 = 0.2mM) than free galactose, clustering of Neu5Ac in oligosaccharides and glycoproteins does not lead to stronger inhibition, which is in contrast to galactose-containing ligands. A more branched (triantennary) sialooligosaccharide inhibits less than biantennary and unbranched sialooligosaccharides. This may be the reason, why complex sialic acid-containing ligands like native orosomucoid or blood cells are not bound and internalized by the macrophages. The dissociation of asialoorosomucoid from the receptor is slow under the influence of Neu5Ac and requires relatively high concentrations of this sugar, whereas the dissociation mediated by galactose is rapid and requires lower concentrations. An allosteric influence of Neu5Ac on the binding of galactose by the receptor is discussed.

Animals↗
PubMed1990

4-Methylumbelliferyl-alpha-glycosides of partially O-acetylated N-acetylneuraminic acids as substrates of bacterial and viral sialidases.

4-O-Acetylated, 7-O-acetylated, and 9-O-acetylated 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acids (Neu4,5Ac2-MU, Neu5,7Ac2-MU, Neu5,9Ac2-MU) were tested as substrates of sialidases of Vibrio cholerae and of Clostridium perfringens. Both sialidases were unable to hydrolyse Neu4,5Ac2-MU. This compound at 1 mM concentration did not inhibit significantly the cleavage of Neu5Ac-MU, the best substrate tested. The 4-O-acetylated sialic acid glycoside is hydrolysed slowly by the sialidase from fowl plague virus. The relative substrate specificity, reflected in V/Km of the Vibrio cholerae sialidase is Neu5Ac-MU much greater than Neu5,7Ac2-MU approximately Neu5,9Ac2-MU and of the clostridial enzyme it is Neu5Ac-MU greater than Neu5,9Ac2-MU greater than Neu5,7Ac2-MU. The affinities of both enzymes for the side-chain O-acetylated sialic acid derivatives are higher than for Neu5Ac-MU. The artificial, well-defined substrates, described here, provide the opportunity to quantify the influence of sialic acid O-acetylation on the hydrolysis of sialoglycoconjugates without the side effects introduced by other parts of more complex glycans.

Acetylation↗