A new UV-B absorbing mycosporine with photo protective activity from the lichenized ascomycete Collema cristatum.

A novel photo protective mycosporine was isolated from the lichenized ascomycete Collema cristatum. Biological activity was measured in terms of protection against UV-B induced membrane destruction and pyrimidine dimer formation in cultured human keratinocytes, and prevention of UV-B induced erythema. It was found that the pure isolated compound prevented UV-B induced cell destruction in a dose-dependent manner, that the compound partially prevented pyrimidine dimer formation and completely prevented UV-B induced erythema when applied to the skin prior to irradiation.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Authors index

In a runoff irrigation system in Northern Kenya, we studied the nutrient interactions of sole cropped and alley cropped Sorghum bicolor (L.) Moench and Acacia saligna (Labill.) H.L. Wendl. The trees were pruned once before the cropping season and the biomass was used as fodder for animals. The nutrient contents in leaf tissue, soil and soil solution were monitored and the uptake of applied tracers (¹⁵N, Sr) was followed. The grain yield of alley cropped sorghum was similar to or slightly higher than in monoculture and did not decrease near the tree-crop interface. Foliar N and Ca contents of the crop were higher in the agroforestry combination than in monoculture, corresponding to higher soil N and Ca contents. Soil solution and soil mineral N dynamics indicate an increase of N under the tree row and unused soil N at the topsoil in the alley of the sole cropped trees as well as below 60 cm depth in the crop monoculture. The N use efficiency of the tree+crop combination was higher than the sole cropped trees or crops. Competition was observed for Zn and Mn of both tree and crop whereas for Ca only the tree contents decreased. P, K, Mg and Fe dynamics were not affected by alley cropping at our site. The lower uptake of applied Sr by trees in alley cropping compared to those of the monoculture stand suggested a lower competitiveness of the acacia than sorghum, which did not show lower Sr contents when intercropped. The study showed the usefulness of combining soil and plant analyses together with tracer techniques identifying nutrient competition, nutrient transfer processes and the complementary use of soil nutrients, as the main features of the tree-crop combination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cthulhu Macrofasciculumque n. g., n. sp. and Cthylla Microfasciculumque n. g., n. sp., a Newly Identified Lineage of Parabasalian Termite Symbionts

The parabasalian symbionts of lower termite hindgut communities are well-known for their large size and structural complexity. The most complex forms evolved multiple times independently from smaller and simpler flagellates, but we know little of the diversity of these small flagellates or their phylogenetic relationships to more complex lineages. To understand the true diversity of Parabasalia and how their unique cellular complexity arose, more data from smaller and simpler flagellates are needed. Here, we describe two new genera of small-to-intermediate size and complexity, represented by the type species Cthulhu macrofasciculumque and Cthylla microfasciculumque from Prorhinotermes simplex and Reticulitermes virginicus, respectively (both hosts confirmed by DNA barcoding). Both genera have a single anterior nucleus embeded in a robust protruding axostyle, and an anterior bundle flagella (and likely a single posterior flagellum) that emerge slightly subanteriorly and have a distinctive beat pattern. Cthulhu is relatively large and has a distinctive bundle of over 20 flagella whereas Cthylla is smaller, has only 5 anterior flagella and closely resembles several other parababsalian genera. Molecular phylogenies based on small subunit ribosomal RNA (SSU rRNA) show both genera are related to previously unidentified environmental sequences from other termites (possibly from members of the Tricercomitidae), which all branch as sisters to the Hexamastigitae. Altogether, Cthulhu likely represents another independent origin of relatively high cellular complexity within parabasalia, and points to the need for molecular characterization of other key taxa, such as Tricercomitus.     

Heat stress responses and population genetics of the kelp Laminaria digitata (Phaeophyceae) across latitudes reveal differentiation among North Atlantic populations

To understand the thermal plasticity of a coastal foundation species across its latitudinal distribution, we assess physiological responses to high temperature stress in the kelp Laminaria digitata in combination with population genetic characteristics and relate heat resilience to genetic features and phylogeography. We hypothesize that populations from Arctic and cold‐temperate locations are less heat resilient than populations from warm distributional edges. Using meristems of natural L. digitata populations from six locations ranging between Kongsfjorden, Spitsbergen (79°N), and Quiberon, France (47°N), we performed a common‐garden heat stress experiment applying 15°C to 23°C over eight days. We assessed growth, photosynthetic quantum yield, carbon and nitrogen storage, and xanthophyll pigment contents as response traits. Population connectivity and genetic diversity were analyzed with microsatellite markers. Results from the heat stress experiment suggest that the upper temperature limit of L. digitata is nearly identical across its distribution range, but subtle differences in growth and stress responses were revealed for three populations from the species’ ecological range margins. Two populations at the species’ warm distribution limit showed higher temperature tolerance compared to other populations in growth at 19°C and recovery from 21°C (Quiberon, France), and photosynthetic quantum yield and xanthophyll pigment responses at 23°C (Helgoland, Germany). In L. digitata from the northernmost population (Spitsbergen, Norway), quantum yield indicated the highest heat sensitivity. Microsatellite genotyping revealed all sampled populations to be genetically distinct, with a strong hierarchical structure between southern and northern clades. Genetic diversity was lowest in the isolated population of the North Sea island of Helgoland and highest in Roscoff in the English Channel. All together, these results support the hypothesis of moderate local differentiation across L. digitata's European distribution, whereas effects are likely too weak to ameliorate the species’ capacity to withstand ocean warming and marine heatwaves at the southern range edge.     

Acceptor substrate specificity of UDP-Gal: GlcNAc-R β1,3-galactosyltransferase (WbbD) from Escherichia coli O7:K1

Most of the glycosyltransferases involved in O antigen biosynthesis have not yet been characterized. We recently demonstrated that the wbbD gene of the O7 lipopolysaccharide biosynthesis cluster in E. coli strain VW187 (O7:K1) encodes WbbD, a UDP-Gal: GlcNAcα-pyrophosphate-lipid β1,3-Gal-transferase (EC 2.4.1., accession number AAC27537) that transfers the second sugar moiety in the assembly of the O7 repeating unit. The enzyme utilizes undecaprenol-pyrophosphate-GlcNAc as a natural acceptor substrate, but can also transfer Gal to GlcNAcα-PO₃-PO₃-(CH₂)₁₁-O-phenyl (GlcNAc-PP-PhU). A number of acceptor substrate analogs have now been tested to further characterize the acceptor specificity of WbbD and to determine the roles of the pyrophosphate bond and the lipid moiety in the acceptor substrate. The enzyme was found to have a low activity with a substrate containing only one phosphate group directly α-linked to GlcNAc, and the enzyme was inactive when the phosphate was absent or further removed from the anomeric carbon of GlcNAc. Modifications of the lipid chain yielded substrates with variable activities. GlcNAc derivatives that were inactive as substrates did not inhibit WbbD suggesting that these compounds did not bind to the active site of the enzyme. The specificity of mammalian β4-galactosyltransferase I has been compared to that of WbbD. The results indicate that the bacterial WbbD enzyme has a distinct specificity for GlcNAc-PP-lipid, and that WbbD recognition of its acceptor substrate is very different from that of the ubiquitous mammalian β4-galactosyltransferase I. These studies help to understand mechanisms of O antigen synthesis, to develop methods to synthesize defined oligosaccharide structures and to develop specific O antigen inhibitors.     

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.     

Genetic studies in south Balkan populations

Within a study of the genetics of Balkan populations, four DNA-STR systems and 19 classical markers were examined in seven samples: Romanians (two groups), Albanians, Greeks and Aromuns (three groups). The results for the DNA-STR systems have been compared with data from the literature. The results show four clear separated groups: sub-Saharan black populations, North-African, Japanese and European populations. The large Balkan populations, except the Greek sample, are genetically more homogenous than the Aromun populations. A second Neighbor-joining tree based on all 23 analyzed systems, show a particular trend of the Aromun groups, which indicates a particular genetic structure.     

Anthrax and the inflammasome

Anthrax lethal toxin (LT), a major virulence determinant of anthrax disease, induces vascular collapse in mice and rats. LT activates the Nlrp1 inflammasome in macrophages and dendritic cells, resulting in caspase-1 activation, IL-1β and IL-18 maturation and a rapid cell death (pyroptosis). This review presents the current understanding of LT-induced activation of Nlrp1 in cells and its consequences for toxin-mediated effects in rodent toxin and spore challenge models.