Anthrax among heroin users in Europe possibly caused by same Bacillus anthracis strain since 2000.

Injection anthrax was described first in 2000 in a heroin-injecting drug user in Norway. New anthrax cases among heroin consumers were detected in the United Kingdom (52 cases) and Germany (3 cases) in 2009-10. In June 2012, a fatal case occurred in Regensburg, Bavaria. As of December 2012, 13 cases had been reported in this new outbreak from Germany, Denmark, France and the United Kingdom. We analysed isolates from 2009-10 and 2012 as well as from the first injection anthrax case in Norway in 2000 by comparative molecular typing using a high resolution 31 marker multilocus variable-number tandem repeat analysis (MLVA) and a broad single nucleotide polymorphism (SNP) analysis. Our results show that all cases may be traced back to the same outbreak strain. They also indicate the probability of a single source contaminating heroin and that the outbreak could have lasted for at least a decade. However, an additional serological pilot study in two German regions conducted in 2011 failed to discover additional anthrax cases among 288 heroin users.

Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.

A soluble form of L-selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L-selectin, recombinantly expressed in baby-hamster kidney cells, were determined. The N-linked glycans were enzymically released and fluorescently labelled with 2-aminobenzamide. Sialylation of the N-glycans was analysed by anion-exchange chromatography followed by rechromatography of the resulting fractions on amino-phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two-dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L-selectin carries bi-, tri- and tetra-antennary sugar chains, which are fucosylated on the innermost residue of N-acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the alpha 2-3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N-acetylgalactosamine, or the H (O) blood-group determinant were also identified. O-Glycosylation of mucin-type sugar chains was not detected in recombinant soluble human L-selectin.

In vivo modulation of the acidic N-glycans from rat liver dipeptidyl peptidase IV by N-propanoyl-D-mannosamine.

Derivatives of N-acyl-D-mannosamine differing in the N-acyl-side chain can be metabolically converted into neuraminic acids with corresponding N-acyl side chains. In the present study we show the in vivo modulation of sialic acids in membrane-bound dipeptidyl peptidase IV (CD 26) from rat liver after administration of N-propanoyl-D-mannosamine. Treatment of rats with this unphysiological precursor resulted in an incorporation of N-propanoylneuraminic acid into N-linked glycans of dipeptidyl peptidase IV.

Cell surface glycoproteins undergo postbiosynthetic modification of their N-glycans by stepwise demannosylation.

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.

Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells.

The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.

O-linked L-fucose is present in Desmodus rotundus salivary plasminogen activator.

DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.

Biosynthetic modulation of sialic acid-dependent virus-receptor interactions of two primate polyoma viruses.

Sialic acids are essential components of the cell surface receptors of many microorganisms including viruses. A synthetic, N-substituted D-mannosamine derivative has been shown to act as precursor for structurally altered sialic acid incorporated into glycoconjugates in vivo (Kayser, H., Zeitler, R., Kannicht, C., Grunow, D., Nuck, R., and Reutter, W. (1992) J. Biol. Chem. 267, 16934-16938). In this study we have analyzed the potential of three different sialic acid precursor analogues to modulate sialic acid-dependent virus receptor function on different cells. We show that treatment with these D-mannosamine derivatives can result in the structural modification of about 50% of total cellular sialic acid content. Treatment interfered drastically and specifically with sialic acid-dependent infection of two distinct primate polyoma viruses. Both inhibition (over 95%) and enhancement (up to 7-fold) of virus binding and infection were observed depending on the N-acyl substitution at the C-5 position of sialic acid. These effects were attributed to the synthesis of metabolically modified, sialylated virus receptors, carrying elongated N-acyl groups, with altered binding affinities for virus particles. Thus, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions and could be a potent tool to further clarify the biological functions of sialic acid, in particular its N-acyl side chain.

Isolation and partial characterization of basic proteinases from stem bromelain.

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine-->serine) and position 20 (asparagine-->glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM, kcat 0.87 sec-1 and F5 (Km 2.42 mM, kcat 0.68 sec-1), and differed greatly from F9 (Km 0.40 mM, kcat 3.94 sec-1).

Synthesis of 2-deoxy-D-galactose containing gangliosides in vivo.

Incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of different gangliosides of rat liver was examined. After intraperitoneal administration of 2-deoxy-D-galactose it was shown by GLC/MS analysis that this hexose analogue is metabolized and incorporated into all the gangliosides investigated, and predominantly into GM3 and GD3. In both of these gangliosides, 25-55% of the galactose residues were substituted by 2-deoxy-D-galactose. The epimer, 2-deoxy-D-glucose, was not detectable.

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.

Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells.

The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.

[Possibilities and limits of daily coping with illness by patients with chronic polyarthritis]. / Möglichkeiten und Grenzen alltäglicher Krankheitsbewältigung durch cP-Patienten.

A central problem of persons with chronic polyarthritis is the necessary adaptation to deteriorating health in everyday activities. Difficulties and burdens of this adaptation are parts of the study undertaken in the Unna Model, which was part of the research on "Local Services for CP-Patients". In the first part two female patients are described with regards to these everyday problems of "normalization", i.e., including health conditions resp. daily activities. An important element is the microsocial context: mutual expectations and support in the family and at the work place. In the second part some quantitative results of interviews with 93 cP-patients are presented. They show the importance that is given by these interviewed persons to these daily adaptations, self help, and support.