Lactotetraosylceramide, a novel glycosphingolipid receptor for Helicobacter pylori, present in human gastric epithelium

The binding of Helicobacter pylori to glycosphingolipids was examined by binding of (35)S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. In addition to previously reported binding specificities, a selective binding to a non-acid tetraglycosylceramide of human meconium was found. This H. pylori binding glycosphingolipid was isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, were identified as Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer (lactotetraosylceramide). When using non-acid glycosphingolipid preparations from human gastric epithelial cells, an identical binding of H. pylori to the tetraglycosylceramide interval was obtained in one of seven samples. Evidence for the presence of lactotetraosylceramide in the binding-active interval was obtained by proton NMR spectroscopy of intact glycosphingolipids and by gas chromatography-electron ionization mass spectrometry of permethylated tetrasaccharides obtained by ceramide glycanase hydrolysis. The lactotetraosylceramide binding property was detected in 65 of 74 H. pylori isolates (88%). Binding of H. pylori to lactotetraosylceramide on thin-layer chromatograms was inhibited by preincubation with lactotetraose but not with lactose. Removal of the terminal galactose of lactotetraosylceramide by galactosidase hydrolysis abolished the binding as did hydrazinolysis of the acetamido group of the N-acetylglucosamine. Therefore, Galbeta3GlcNAc is an essential part of the binding epitope.     

Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters

BACKGROUND: The lipopolysaccharide of Helicobacter pylori plays an important role in colonization and pathogenicity. The present study sought to compare structural and biological features of lipopolysaccharides from gastric and enterohepatic Helicobacter spp. not previously characterized. MATERIALS AND METHODS: Purified lipopolysaccharides from four gastric Helicobacter spp. (H. pylori, Helicobacter felis, Helicobacter bizzozeronii and Helicobacter mustelae) and four enterohepatic Helicobacter spp. (Helicobacter hepaticus, Helicobacter bilis, 'Helicobacter sp. flexispira' and Helicobacter pullorum) were structurally characterized using electrophoretic, serological and chemical methods. RESULTS: Structural insights into all three moieties of the lipopolysaccharides, i.e. lipid A, core and O-polysaccharide chains, were gained. All species expressed lipopolysaccharides bearing an O-polysaccharide chain, but H. mustelae and H. hepaticus produced truncated semirough lipopolysaccharides. However, in contrast to lipopolysaccharides of H. pylori and H. mustelae, no blood group mimicry was detected in the other Helicobacter spp. examined. Intra-species, but not interspecies, fatty acid profiles of lipopolysaccharides were identical within the genus. Although shared lipopolysaccharide-core epitopes with H. pylori occurred, differing structural characteristics were noted in this lipopolysaccharide region of some Helicobacter spp. The lipopolysaccharides of the gastric helicobacters, H. bizzozeronii and H. mustelae, had relative Limulus amoebocyte lysate activities which clustered around that of H. pylori lipopolysaccharide, whereas H. bilis, 'Helicobacter sp. flexispira' and H. hepaticus formed a cluster with approximately 1000-10,000-fold lower activities. H. pullorum lipopolysaccharide had the highest relative Limulus amoebocyte lysate activity of all the helicobacter lipopolysaccharides (10-fold higher than that of H. pylori lipopolysaccharide), and all the lipopolysaccharides of enterohepatic Helicobacter spp. were capable of inducing nuclear factor-Kappa B(NF-kappaB) activation. CONCLUSIONS: The collective results demonstrate the structural heterogeneity and pathogenic potential of lipopolysaccharides of the Helicobacter genus as a group and these differences in lipopolysaccharides may be indicative of adaptation of the bacteria to different ecological niches.     

A link between Helicobacter pylori and/or Chlamydia spp. infections and atherosclerosis

Antibodies to Helicobacter pylori, Chlamydia spp. and Mycobacterium bovis were determined in patients with coronary heart disease, H. pylori-related dyspepsia, and tuberculosis, and healthy controls. Enzyme-linked immunosorbent assay was conducted with a glycine extract and CagA protein of H. pylori, chlamydial lipopolysaccharide and mycobacterial heat shock protein Hsp65. The prevalence of anti-glycine extract IgG in coronary heart disease patients was higher than in the tuberculosis group and controls, and the same as in dyspeptic patients. Anti-chlamydial IgG were more prevalent in the coronary heart disease group than in healthy subjects. There was no difference in the prevalence of anti-CagA IgG in the coronary heart disease group and controls or anti-Hsp65 IgG in the patients with coronary heart disease, dyspepsia, tuberculosis, and controls. Anti-glycine extract IgA (like anti-glycine extract IgG) were more prevalent in the coronary heart disease group than in the healthy group. The highest anti-glycine extract IgG/IgA and anti-chlamydial IgG titers were more frequent in coronary heart disease patients as compared with controls. Infections with H. pylori and Chlamydia spp. and enhanced production of antibodies to these pathogens may predispose to human atherosclerosis.     

Protein kinase A intersects SRC signaling in membrane microdomains

Regulation of Src kinase activity is tightly coupled to the phosphorylation status of the C-terminal regulatory tyrosine Tyr(527), which, when phosphorylated by Csk, represses Src. Here, we demonstrate that activation of Csk through a prostaglandin E(2)-cAMP-protein kinase A (PKA) pathway inhibits Src. This inhibitory pathway is operative in detergent-resistant membrane fractions where cAMP-elevating agents activate Csk, resulting in a concomitant decrease in Src activity. The inhibitory effect on Src depends on a detergent-resistant membrane-anchored Csk and co-localization of all components of the inhibitory pathway in membrane microdomains. Furthermore, epidermal growth factor-induced activation of Src and phosphorylation of the Src substrates Cbl and focal adhesion kinase are inhibited by activation of the cAMP-PKA-Csk pathway. We propose a novel mechanism whereby G protein-coupled receptors inhibit Src signaling by activation of Csk in a cAMP-PKA-dependent manner.     

Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure

Sequence comparisons and a revised classification of the Euglenophyceae were based on 92 new SSU rDNA sequences obtained from strains of Euglena, Astasia, Phacus, Trachelomonas, Colacium, Cryptoglena, Lepocinclis, Eutreptia, Eutreptiella and Tetreutreptia. Sequence data also provided molecular signatures for taxa from genus to class level in the SSU rRNA secondary structure, revealed by a novel approach (search for non-homoplasious synapomorphies) and used for taxonomic diagnoses. Photosynthetic euglenoids and secondary heterotrophs formed a clade, designated as Euglenophyceae (emend.) with two orders: Euglenales and Eutreptiales. The mostly marine Eutreptiales (Eutreptia, Eutreptiella; not Distigma) comprised taxa with two or four emergent flagella (the quadriflagellate Tetreutreptia was integrated within Eutreptiella). The Euglenales (freshwater genera with < or = one emergent flagellum) formed nine clades and two individual branches (single strains); however, only two clades were congruent with traditional genera: Trachelomonas (incl. Strombomonas) and Colacium. Euglena was polyphyletic and diverged into four independent clades (intermixed with Astasia, Khawkinea and Lepocinclis) and two individual branches (e.g. E. polymorpha). Phacus was also subdivided into Phacus s. str. and two combined lineages (mixed with Lepocinclis spp. or Cryptoglena). In consequence, Euglena (s. str.), Phacus and other genera were emended and one lineage (mixed Phacus/Lepocinclis-clade) was recognized as the previously neglected genus Monomorphina Mereschkowsky (1877). The sister clade of Phacus s. str. (mixed Euglena/Lepocinclis-clade) was identified as Lepocinclis Perty (emended).     

A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Abstract Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli β-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Increased metal tolerance in Salix by nicotinamide and nicotinic acid

We have earlier shown that nicotinamide (NIC) and nicotinic acid (NiA) can induce defence-related metabolism in plant cells; e.g. increase the level of glutathione. Here we investigated if NIC and NiA could increase the metal tolerance in metal sensitive clones of Salix viminalis and whether this would be mediated via increased glutathione level. Salix clones, sensitive or tolerant to zinc (Zn), copper (Cu) and cadmium (Cd) were grown in the presence of heavy metals (Cd, Cu or Zn) or NIC and NiA as well as in combination. In addition, the influence of N-acetyl-cystein (NAC) and l-2-oxothiazolidine 4-carboxylate (OTC), stimulators of reduced glutathione (GSH) biosynthesis, and the glutathione biosynthesis inhibitor buthionine sulfoximine (BSO) was analysed. Tolerance was measured as effects on root and shoot dry weight, and the glutathione and metal concentrations in the tissues were analysed. Results showed that NIC and NiA decreased the toxic effects of Cd, Cu and Zn on growth significantly in sensitive clones, but also to some extent in tolerant clones. However, the glutathione level and metal concentration did not change by NIC or NiA addition. Treatment with NAC, OTC or BSO did not per se influence the sensitivity to Cd, although the glutathione level increased in the presence of NAC and OTC and decreased in response to BSO. The results suggest that NIC and NiA increased the defence against heavy metals but not via glutathione formation per se.