Synthesis of a disaccharide fragment of rhamnogalacturonan II

A disaccharide portion of the A-side chain of the rhamnogalacturonan II oligosaccharide has been prepared. Glycosylation of methyl (methyl 3,4-O-isopropylidene-alpha-D-galactopyranosid)uronate with p-tolyl 2,3-di-O-acetyl-3-C-(benzyloxymethyl)-1-thio-alpha/beta-D-erythrofuranoside was carried out using N-iodosuccinimide as promoter and silver trifluoromethanesulfonate as catalyst. Removal of the protecting groups gave the beta-d-Apif-(1-->2)-alpha-D-GalpA-OMe disaccharide.     

Regulation of the DNA replication fork: a way to fight genomic instability

DNA replication is a complex mechanism that functions due to the coordinated interplay of many factors. In the last few years, numerous studies have suggested that DNA replication factors are closely implicated in several DNA transaction events that maintain the integrity of the genome. Therefore, DNA replication fork factors have to be considered as part of a general process that aims to protect and replicate the genome in order to allow correct functioning of a cell and its eventual daughter cells. This is illustrated by the numerous factors that have a well-defined function at the DNA replication fork, but also play crucial roles in different DNA repair pathways such as base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair. Moreover, several of the replisome proteins have also been shown to be essential in sensing and transducing DNA damages through the checkpoint cascade pathways, including the recently characterised alternative clamps and clamp-loaders. In this review we present DNA replication factors that are involved in different DNA transaction and checkpoint regulation pathways, with emphasis on the link between DNA replication and maintenance of genomic stability.     

Evidence of finely tuned expression of DNA polymerase beta in vivo using transgenic mice

DNA polymerase (Pol) is an error-prone repair DNA polymerase that has been shown to create genetic instability and tumorigenesis when overexpressed by only 2-fold in cells, suggesting that a rigorous regulation of its expression may be essential in vivo. To address this question, we have generated mice which express a transgene (Tg) bearing the Pol cDNA under the control of the ubiquitous promoter of the mouse H-2K gene from the major histocompatibility complex. These mice express the Tg only in thymus, an organ which normally contains the most abundant endogenous Pol mRNA and protein, supporting the idea of a tight regulation of Pol in vivo. Furthermore, we found no tumor incidence, suggesting that the single Pol overexpression event is not sufficient to initiate tumorigenesis in vivo.     

Evidence for genetic control of adult weight plasticity in the snail Helix aspersa

Phenotypic plasticity and canalization are important topics in quantitative genetics and evolution. Both concepts are related to environmental sensitivity. The latter can be modeled using a model with genetically structured environmental variance. This work reports the results of a genetic analysis of adult weight in the snail Helix aspersa. Several models of heterogeneous variance are fitted using a Bayesian, MCMC approach. Exploratory analyses using posterior predictive model checking and model comparisons based on the deviance information criterion favor a model postulating a genetically structured heterogeneous environmental variance. Our analysis provides a strong indication of a positive genetic correlation between additive genetic values affecting the mean and those affecting environmental variation of adult body weight. The possibility of manipulating environmental variance by selection is illustrated numerically using estimates of parameters derived from the snail data set.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

External membrane vesicles from Helicobacter pylori induce apoptosis in gastric epithelial cells

The Helicobacter pylori infection of gastric mucosa is one of the most common infectious diseases and is associated with a variety of clinical outcomes, including peptic ulcer disease and gastric cancer. Helicobacter pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors, which include VacA and CagA. Outer membrane vesicles are constantly shed by the bacteria and can provide an additional mechanism for pathogenicity by releasing non-secretable factors which can then interact with epithelial cells. The present report shows that external membrane vesicles are able to induce apoptosis not mediated by mitochondrial pathway in gastric (AGS) epithelial cells, as demonstrated by the lack of cytochrome c release with an activation of caspase 8 and 3. Apoptosis induced by these vesicles does not require a classic VacA+ phenotype, as a negative strain with a truncated and therefore non-secretable form of this protein can also induce cell death. These results should be taken into account in future studies of H. pylori pathogenicity in strains apparently VacA-.     

Pneumocystis oryctolagi sp. nov., an uncultured fungus causing pneumonia in rabbits at weaning: review of current knowledge, and description of a new taxon on genotypic, phylogenetic and phenotypic bases

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.