Fiber-degrading systems of different strains of the genus Fibrobacter

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.     

Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

Central assumptions of predator-prey models fail in a semi-natural experimental system

The relationship between the encounter rate of predators with prey and the density of this prey is fundamental to models of predator-prey interactions. The relationship determines, among other variables, the rate at which prey patches are depleted, and hence the impact of predator populations on their prey, and the optimal spatial distribution of foraging effort. Two central assumptions that are made in many models are that encounter rate is directly proportional to prey density and that it is independent of the proportion of prey already removed, other than via the decreased density. We show here, using captive great tits searching for winter moth caterpillars in their natural hiding positions, that neither of these assumptions hold. Encounter rate increased less than directly in proportion to prey density, and it depended not only on the current density of prey, but also on the proportion of prey already removed by previous foragers. Both of these effects are likely to have major consequences for the outcome of predator-prey interactions.     

Single-chain Tet transregulators

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.     

Impact of DNA ligase IV on the fidelity of end joining in human cells

A DNA ligase IV (LIG4)-null human pre-B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error-prone DNA ligase IV-independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining. Thus, DNA ligase IV is an important factor influencing the fidelity of end joining in vivo. The LIG4-defective cell lines also showed impaired end joining in an in vitro assay using cell-free extracts. Elevated degradation of the terminal nucleotide was observed in a LIG4-defective line, and addition of the DNA ligase IV–XRCC4 complex restored end protection. End protection by DNA ligase IV was not dependent upon ligation. Finally, using purified proteins, we demonstrate that DNA ligase IV–XRCC4 is able to protect DNA ends from degradation by T7 exonuclease. Thus, the ability of DNA ligase IV–XRCC4 to protect DNA ends may contribute to the ability of DNA ligase IV to promote accurate rejoining in vivo.     

Oxidant-induced DNA damage by quartz in alveolar epithelial cells

Respirable quartz has recently been classified as a human carcinogen. Although, studies with quartz using naked DNA as a target suggest that formation of oxyradicals by particles may play a role in the DNA-damaging properties of quartz, it is not known whether this pathway is important for DNA damage in the target cells for quartz carcinogenesis, i.e. alveolar epithelial cells. Therefore, we determined in vitro DNA damage by DQ12 quartz particles in rat and human and alveolar epithelial cells (RLE, A549) using the single cell gel electrophoresis/comet assay. The radical generation capacity of quartz was analysed by electron spin resonance (ESR) and by immunocytochemical analysis of the hydroxyl radical-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the epithelial cells. Quartz particles as well as the positive control hydrogen peroxide, caused a dose-dependent increase in DNA strand breaks in both cell lines. DNA damage by quartz was significantly reduced in the presence of the hydroxyl-radical scavengers mannitol or DMSO. The involvement of hydroxyl radicals was further established by ESR measurements and was also demonstrated by the ability of the quartz to induce formation of 8-OHdG. In conclusion, our data show that quartz elicits DNA damage in rat and human alveolar epithelial cells and indicate that these effects are driven by hydroxyl radical-generating properties of the particles.     

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.