The miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site

5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C → T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC ~5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.     

Phylogenetic analysis of bacterial populations in waters of the former Texcoco Lake, Mexico

Molecular techniques were used to compare the compositions of the bacterial communities of the 2 following lagoons from the former soda Texcoco Lake, Mexico: the restored Facultativa lagoon and the Nabor Carrillo lagoon. Ribosomal intergenic spacer analysis (RISA) revealed that bacterial communities of the 2 lagoons were different and presented a relatively low diversity. Clone libraries of 16S rDNA genes were constructed, and significant phylotypes were distinguished by restriction fragment length polymorphism (RFLP). A representative clone from each phylotype was partially sequenced. Molecular identification and phylogenetic analyses based on ribosomal sequences revealed that the Facultativa lagoon harbored mainly gamma- and beta-Proteobacteria, low G+C Gram-positive bacteria, and several members of the Halobacteriaceae family of archaea. The Nabor Carrillo lagoon mainly included typical halophilic and alkaliphilic low G+C Gram-positive bacteria, gamma-Proteobacteria, and beta-Proteobacteria similar to those found in other soda lakes. Several probably noncultured new bacterial species were detected. Three strains were isolated from the Nabor Carrillo lagoon, their partial 16S rDNA sequences were obtained. On this basis, they were identified as Halomonas magadiensis (H1), Halomonas eurihalina (H2), and Staphylococcus sciuri (H3). This is the first study that uses molecular techniques to investigate potential genetic diversity in the Texcoco lakes. In this preliminary evaluation, we infer the presence of alkalophilic, halophilic, or haloalkaliphilic bacteria potentially useful for biotechnology.     

A Universal Model of Commuting Networks

We show that a recently proposed model generates accurate commuting networks on 80 case studies from different regions of the world (Europe and United-States) at different scales (e.g. municipalities, counties, regions). The model takes as input the number of commuters coming in and out of each geographic unit and generates the matrix of commuting flows between the units. The single parameter of the model follows a universal law that depends only on the scale of the geographic units. We show that our model significantly outperforms two other approaches proposing a universal commuting model [1], [2], particularly when the geographic units are small (e.g. municipalities).     

<Emphasis Type="Italic">Geosmithia</Emphasis> Fungi are Highly Diverse and Consistent Bark Beetle Associates: Evidence from their Community Structure in Temperate Europe

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997–2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species’ niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species’ co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect–fungus association, contrary to previous hypotheses. An erratum to this article can be found at     

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

Caught bending the A-rule: crystal structures of translesion DNA synthesis with a non-natural nucleotide

Damage to DNA involving excision of the nucleobase at the N-glycosidic bond forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired abasic site during DNA synthesis, most B family polymerases obey the A-rule and preferentially incorporate dAMP without instruction from the template. In addition to being potentially mutagenic, abasic sites provide strong blocks to DNA synthesis. A previous crystal structure of an exonuclease deficient variant of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43 exo-) illustrated these properties, showing that the polymerase failed to translocate the DNA following insertion of dAMP opposite an abasic site. We examine four new structures depicting several steps of translesion DNA synthesis by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP opposite abasic sites. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP. As with dAMP, the DNA fails to translocate following insertion of 5-NIMP, although distortions at the nascent primer terminus contribute less than previously thought in inducing the stall, given that 5-NIMP preserves relatively undistorted geometry at the insertion site following phosphoryl transfer. An open ternary configuration, novel in B family polymerases, reveals an initial template independent binding of 5-NITP adjacent to the active site of the open polymerase, suggesting that closure of the fingers domain shuttles the nucleotide to the active site while testing the substrate against the template.     

Development of antibodies against secoisolariciresinol--application to the immunolocalization of lignans in Linum usitatissimum seeds

Lignans are widely distributed plant metabolites associated with a large range of biological activities. In order to gain insight into their biosynthesis and their spatio-temporal accumulation an immunological probe was developed. Secondary metabolites generally have too small molecular weight to be antigenic and have to be associated with a carrier protein. Secoisolariciresinol was chosen as the hapten and was linked to bovine serum albumin via a spacer arm, the p-aminohippuric acid. The artificial antigen was injected to New Zealand rabbits. The successful production of polyclonal antibodies against secoisolariciresinol was assessed with indirect enzyme immunosorbent assay (ELISA) by comparison with pre-immune serum and by competitive assays using dilutions of secoisolariciresinol standards. The antibodies had an IC(50) value of 94 μg/ml and showed moderate cross-reactivities with structurally related compounds. They were thus used to immunolocalize lignans in flaxseed (Linum usitatissimum), one of the richest sources of lignans. The immunohistochemical labeling allowed us to localize for the first time lignans in planta. They are mainly localized in the secondary wall of the sclerite cells of the outer integument of the seed. A very light labeling is also observed in cytoplasmic inclusions of the endosperm. The results were correlated with HPLC analytical results which enabled to evaluate the relative lignan quantities: in flaxseed about 90% of the metabolites are localized in the outer integument.     

Biotransformation of natural compounds. Oxido-reduction of Sch-642305 by Aspergillus ochraceus ATCC 1009

Sch-642305 is the major compound produced by the endophytic fungi Phomopsis sp. CMU-LMA. Incubation of Sch-642305 with Aspergillus ochraceus ATCC 1009 resting cells leads to three new derivatives through an oxido-reduction of the six-membered ring of the molecule. Reduction of the double bound leads to compound (1), which subsequently undergoes carbonyl reduction to (2) and ring hydroxylation to (3). According to the previously solved crystal structure of Sch-642305 coupled with ¹H NMR NOE correlation and the crystal structure of compound 1, the absolute configurations of the new derivatives were established. In contrast to the parent compound Sch-642305, compound (1) exhibits antimicrobial activity against Gram-negative bacteria. Furthermore, while all derivatives exhibit cytotoxic activity against various cancer cell lines, compound (2) achieved an IC₅₀ of 4 nM against human myelogenous leukemia K 562, compared to 20 nM for the parent Sch-642305.