Kinetic analysis of the coordinated interaction of SgrAI restriction endonuclease with different DNA targets

SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG, and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (Ka = 4-8 x 10(10) M-1) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by kcat/K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes, which differ in their catalytic efficiencies.     

Potential signalling pathways underlying corticotrophin-releasing hormone-mediated neuroprotection from excitotoxicity in rat hippocampus

In several neurological disorders including cerebral ischaemia, glutamate has been implicated as a neurotoxic agent in the mechanisms leading to neuronal cell death. The role of corticotrophin-releasing hormone (CRH), the 41-amino acid peptide, which activates the HPA axis in response to stressful stimuli, remains controversial. In this study, we report that CRH in low physiological concentrations (2 pM), prevented glutamate-induced neurotoxicity via receptor-mediated mechanisms when administered to organotypic hippocampal cultures both during and after the glutamate-induced insult. Detailed investigations on the mechanisms mediating this neuroprotective effect showed that activation of the adenylate cyclase pathway and induction of MAP kinase phosphorylation mediate the CRH action. In addition we showed that CRH can inhibit the phosphorylation of JNK/SAPK by glutamate. Most importantly, we showed that CRH can afford neuroprotection against neurotoxicity up to 12 h following the insult, suggesting that CRH is acting at a late stage in the neuronal death cycle, and this might be important in the development of novel neuroprotective agents in order to improve neuronal survival following the insult.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.     

The effect of electrodialytic treatment and Na2H2EDTA addition on methanogenic activity of copper-amended anaerobic granular sludge: treatment costs and energy consumption

The effect of electrodialytic treatment in terms of a current density, pH and Na₂H₂EDTA addition on the methanogenic activity of copper-amended anaerobic granular sludge taken from the UASB reactor from paper mill was evaluated. Moreover, the specific energy consumption and simplified operational and treatment costs were calculated. Addition of Na₂H₂EDTA (at pH 7.7) to copper-amended sludge resulted in the highest microbial activity (62 mg CH₄-COD g VSS⁻¹ day⁻¹) suggesting that Na₂H₂EDTA decreased the toxic effects of copper on the methanogenic activity of the anaerobic granular sludge. The highest methane production (159 %) was also observed upon Na₂H₂EDTA addition and simultaneous electricity application (pH 7.7). The energy consumption during the treatment was 560, 840, 1400 and 1680 kW h m⁻³ at current densities of 0.23, 0.34, 0.57 and 0.69 mA cm⁻², respectively. This corresponded to a treatment costs in terms of electricity expenditure from 39.2 to 117.6 € per cubic meter of sludge.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

Preparation, stability and antimicrobial activity of cationic cross-linked starch-iodine complexes

Cationic cross-linked starch (CCS)-iodine complexes containing different amounts of quaternary ammonium groups (different degrees of substitution (DS)) and iodine have been obtained by iodine adsorption on CCS from aqueous iodine potassium iodide solution. Equilibrium adsorption studies showed that with an increase of DS the amount of iodine adsorbed on CCS and the affinity of iodine to CCS increased linearly. The influences of the DS of CCS and the amount of adsorbed iodine on the stability of CCS-iodine complexes in a solution of 0.02M sodium acetate and reactivity toward l-tyrosine have been investigated. At the same DS, the stability of CCS-iodine complexes decreased with an increase of the amount of adsorbed iodine. With increasing the DS, the stability of CCS-iodine complexes increased. The iodine consumption in the reaction with l-tyrosine increased significantly with an increase of the amount of adsorbed iodine. The influence of DS on iodine consumption was lower and depended on the amount of adsorbed iodine. The antibacterial activity of CCS-iodine complexes against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was determined by the broth-dilution and spread-plate methods. The obtained results have demonstrated that an appropriate selection of the CCS-iodine complex composition (the DS of CCS and the amount of adsorbed iodine) could ensure good antimicrobial properties by keeping a low concentration of free iodine in the system. The main advantage of using CCS-iodine complexes as antimicrobial agents is the biodegradability of the polymeric matrix.     

Genus delimitation,biogeography and diversification of Choristoneura Lederer (Lepidoptera: Tortricidae) based on molecular evidence

Widely known for pest species that include major modulators of temperate forests, the genus Choristoneura is part of the species‐rich tribe Archipini of leafroller moths (Tortricidae). Delimitation of the genus has remained unresolved because no phylogeny has included species endemic to Africa and studies have often omitted the type species of the genus. Further taxonomic confusion has been generated by the transfer of Archips occidentalis (Walsingham) to Choristoneura, creating a homonym with Choristoneura occidentalis Freeman, an important defoliator of North American forests. To define the limits of the genus, we reconstructed a phylogeny using DNA sequences for mitochondrial cytochrome oxidase subunit I and nuclear ribosomal 28S genes. Our ingroup included 23 Choristoneura species‐level taxa, complemented by a large sample of outgroups comprising 82 species of Archipini and other Tortricidae. We generated a time‐calibrated tree using fossil and secondary calibrations and we inferred biogeographic and diversification processes in Choristoneura. Our analysis recovered the genus as polyphyletic, with Archips occidentalis, Choristoneura simonyi and Choristoneura evanidana excluded from the main clade. Based on the recovered phylogenies and a redefinition, we restrict Choristoneura primarily to species with a northern hemisphere distribution. Our analysis supports A. occidentalis as the sister group of Cacoecimorpha pronubana, C. simonyi as the sister of ‘Xenotemna’ pallorana, and C. evanidana as the sister of Archips purpurana. A new combination is proposed: Archips evanidana comb.n. ; the availability of ‘Xenotemna’ as a valid name is discussed and A. occidentalis is considered as an orphaned name within the Archipini. We found support for a Holarctic origin of Choristoneura about 23 Ma, followed by early divergence in the Palearctic region. The main divergence occurred at 16 Ma, with one clade in the Nearctic and another in the Palearctic. Subsequent cladogenetic events were synchronous and related to herbivorous specialization, with each clade divided into coniferophagous and polyphagous lineages. Their specialization as conifer feeders temporally matched the expansion of boreal forest during the Miocene.     

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.