Blazeispirane and protoblazeispirane derivatives from the cultured mycelia of the fungus Agaricus blazei

Two blazeispirane derivatives including blazeispirols G and I were isolated from the cultured mycelia of the fungus Agaricus blazei Murill and were established to be (20S, 22S, 23R, 24S)-14 beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-triene-11 alpha,23-diol and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-23,28-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A. Furthermore, four blazeispirol derivatives blazeispirols, U, V, V(1) and Z(1) were isolated form the same source described above. Their structures were determined to be (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxyergosta-4,6,8,11-tetraen-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 alpha,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 beta,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxy-4,5-seco-ergosta-6,8-diene-3,5-dione by extensive 1 D and 2D NMR spectral data.     

Abietane diterpenoids from suspension cultured cells of Torreya nucifera var. radicans

Three abietane diterpenoids were isolated from the suspension cultured cells of Torreya nucifera var. radicans along with four known abietane diterpenoids. Based on spectroscopic evidence, the structures of the three were elucidated as (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11-diol, (3S,5R,10S)-7-oxo-12-methoxyabieta-8,11,13-triene-3,11,14-triol and (5R,10S)-3-oxo-7R,12-dimethoxyabieta-8,11,13-trien-11-ol, respectively.     

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.     

IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.     

Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains

Helicobacter pylori, bacteria that colonize the human gastric mucosa, possess a large number of genes for restriction-modification (R-M) systems, and essentially, every strain possesses a unique complement of functional and partial R-M systems. Nearly half of the H.pylori strains studied possess an active type IIs R-M system, HpyII, with the recognition sequence GAAGA. Recombination between direct repeats that flank the R-M cassette allows for its deletion whereas strains lacking hpyIIRM can acquire this cassette through natural transformation. We asked whether strains lacking HpyII R-M activity can acquire an active hpyIIRM cassette [containing a 1.4 kb kanamycin resistance (aphA) marker], whether such acquisition is DNase sensitive or resistant and whether restriction barriers limit acquisition of chromosomal DNA. Our results indicate that natural transformation and conjugation-like mechanisms may contribute to the transfer of large (4.8 kb) insertions of chromosomal DNA between H.pylori strains, that inactive or partial R-M systems can be reactivated upon recombination with a functional allele, consistent with their being contingency genes, and that H.pylori R-M diversity limits acquisition of chromosomal DNA fragments of ≥1 kb.     

Phenotypic and genotypic variation in methylases involved in type II restriction-modification systems in Helicobacter pylori

To determine relationships between Helicobacter pylori geographical origin and type II methylase activity, we examined 122 strains from various locations around the world for methylase expression. Most geographic regions possessed at least one strain resistant to digestion by each of 14 restriction endonucleases studied. Across all of the strains studied, the average number of active methylases was 8.2 ± 1.9 with no significant variation between the major geographic regions. Although seven pairs of isolates showed the same susceptibility patterns, their cagA/vacA status differed, and the remaining 108 strains each possessed unique patterns of susceptibility. From a single clonal group, 15 of 18 strains showed identical patterns of resistance, but diverged with respect to M.MboII activity. All of the methylases studied were present in all major human population groupings, suggesting that their horizontal acquisition pre-dated the separation of these populations. For the hpyV and hpyAIV restriction-modification systems, an in-depth analysis of genotype, indicating extensive diversity of cassette size and chromosomal locations regardless of the susceptibility phenotype, points toward substantial strain-specific selection involving these loci.     

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.