Induction of base damages representing a high risk site for double-strand DNA break formation in genomic DNA by exposure of cells to DNA damaging agents

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.     

Caenorhabditis elegans as a simple model to study phenotypic and genetic virulence determinants of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause disease by invading normally sterile niches within the host body, e.g., urinary tract, blood and cerebrospinal fluid. Infections due to ExPEC strains, in particular urinary tract infections, cause considerable morbidity and significant health-care costs. The goal of our study is to evaluate whether Caenorhabditis elegans can be used as a model to study phenotypic and genetic virulence determinants of ExPEC strains. For this purpose, we used a collection of 31 E. coli strains isolated during acute extra-intestinal infections or from the feces of healthy individuals. For all strains, the phylogeny, the presence of ExPEC virulence factors, the resistance to biologically relevant stressors (bile, human serum and lysozyme), the motility, the growth rate, the virulence in C. elegans and in a murine septicaemia model has been established. The results show that there is a strong link between virulence in C. elegans and certain phenotypic and genetic virulence predictors of ExPEC strains determinable in vitro. Furthermore, there is a significant correlation between virulence of different ExPEC strains in C. elegans and in the murine model. Therefore, our results suggest that C. elegans can be used as a model to study virulence determinants of ExPEC strains.     

Molecular Evidence for the Polyphyly of Macrotrichomonas (Parabasalia: Cristamonadea) and a Proposal for Macrotrichomonoides n. gen.

Macrotrichomonas (Cristamonadea: Parabasalia) is an anaerobic, amitochondriate flagellate symbiont of termite hindguts. It is noteworthy for being large but not structurally complex compared with other large parabasalians, and for retaining a structure similar in appearance to the undulating membrane (UM) of small flagellates closely related to cristamonads, e.g. Tritrichomonas. Here, we have characterised the SSU rDNA from two species described as Macrotrichomonas: M. restis Kirby 1942 from Neotermes jouteli and M. lighti Connell 1932 from Paraneotermes simplicicornis. These species do not form a clade: M. lighti branches with previously characterised Macrotrichomonas sequences from Glyptotermes, while M. restis branches with the genus Metadevescovina. We examined the M. restis UM by light microscopy, scanning electron microscopy, and transmission electron microscopy, and we find common characteristics with the proximal portion of the robust recurrent flagellum of devescovinids. Altogether, we show the genus Macrotrichomonas to be polyphyletic and propose transferring M. restis to a new genus, Macrotrichomonoides. We also hypothesise that the macrotrichomonad body plan represents the ancestral state of cristamonads, from which other major forms evolved.     

Characterization of the cryptic Escherichia lineages: rapid identification and prevalence

Strains phenotypically indistinguishable from Escherichia coli and belonging to at least five distinct cryptic lineages, named Escherichia clades I to V, that are genetically divergent from E. coli yet members of the genus have been recently found using multi-locus sequence typing (MLST). Very few epidemiological data are available on these strains as their detection by MLST is not suitable for large-scale studies. In this work, we developed a rapid PCR method based on aes and chuA allele-specific amplifications that assigns a strain a cryptic lineage membership. By screening more than 3500 strains with this approach, we show that the cryptic lineages of Escherichia are unlikely to be detected in human faecal samples (2-3% frequency) and even less likely to be isolated from extra-intestinal body sites (< 1% frequency). They are more abundant in animal faeces ranging from 3-8% in non-human mammals to 8-28% in birds. Overall, the strains from the clade V are the most abundant and from the clade II very rare. These results suggest that members of the cryptic clades are unlikely to be of significance to human and health but may influence the use of 'E. coli' as an indicator of water quality.     

Metabolic profiling of ethephon-treated sweet cherry (Prunus avium L.)

Increasing costs and decreasing labor availability for sweet cherry harvest in Washington State, USA, has reinvigorated commercial and research interest of mechanized harvest. Ethephon (2-chloroethyl phosphonic acid) can be used to improve fruit abscission for mechanical harvest. Our previous work shows that 3.5 l ha⁻¹ ethephon enhances red color and reduces firmness of the cultivar ‘Bing’. In the current study, we used metabolic profiling of cultivars ‘Bing’, Chelan’, and ‘Skeena’ fruit meso and exocarp tissue to better understand underlying quality-related metabolism associated with ethephon application. Trees were treated using air-blast sprayer 13–14 days prior to harvest and fruit samples were harvested every 7–10 days starting at least 17 days prior to commercial harvest. Nearly 200 identified and partially characterized metabolites from mesocarp and exocarp tissue were characterized and evaluated. Principal component analysis models revealed changes in the metabolome associated with both natural ripening and ethephon-induced changes, including associations to key color, acid, and sugar components, such as cyanidin 3-glucoside, malic acid and sugar metabolism.     

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).     

Termite (Insecta: Isoptera) Species Composition in a Primary Rain Forest and Agroforests in Central Amazonia

Termites play important roles in organic matter decomposition, nutrient cycling, and soil structure in tropical rain forests. When forests are replaced by agriculture, termite species richness, abundance, and function often decline. We compared the termite assemblage of a primary forest site with that of a low plant diversity, palm-based agroforest (five plant species) and a high plant diversity, home-garden agroforest (10 plant species) using a rapid biodiversity assessment protocol. In comparing the primary forest termite species composition to previously published studies, we found soil feeders and the Apicotermitinae to be more dominant than previously reported in Amazonia. Thirty percent of the species belonged to the Apicotermitinae, and an unusually high percentage (57%) of species were soil feeders. Unexpectedly, the palm-based agroforest, despite its lower plant diversity, was closer to primary forest in termite species composition, rate of species accumulation, and proportions of species in taxonomic and functional classes than was the home-garden agroforest. This suggests that particular plant attributes may better determine the termite assemblage than plant diversity alone in these agroecosystems. Unlike other agroecosystems reported in the literature, Apicotermitinae and soil feeders were proportionally more abundant in these agroforests than in primary forest. The ability of agroforests to support populations of soil feeders has a potentially positive effect on soil fertility in these agroecosystems; insomuch as feeding guild is a proxy for function, these closed-canopy agroforests may be able to sustain the same termite-mediated functions as primary forest.     

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.