Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: New features in an amidohydrolase family member

The recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been characterised and its crystal structure elucidated at 1.85 Å. The global architecture of the protein is reminiscent of that of the amidohydrolase superfamily, consisting of two domains; an (α/β)₈ TIM-like barrel domain, where the catalytic centre is located, and a smaller β-sheet sandwich domain of unknown function. The c-terminal tails of each subunit extend toward another monomer in a swapping-like manner, creating a hydrogen bond network which suggests its implication in protein oligomerisation. Mutational and structural evidence suggest the involvement of a conserved tyrosine in the reaction mechanism of the enzyme. SmelDhp presents both hydantoinase and dihydropyrimidinase activities, with higher affinity for the natural six-membered ring substrates. For the five-membered ring substrates, affinity was greater for those with aliphatic and apolar groups in the 5th carbon atom, with the highest rates of hydrolysis for d-5-methyl and d-5-ethyl hydantoin (k_cat/K_m = 2736 ± 380 and 944 ± 52 M^?1 s^?1, respectively). The optimal conditions for the enzyme activity were found to be 60 °C of temperature at pH 8.0. SmelDhp retains 95% of its activity after 6-hour preincubation at 60 °C. This is the first dihydropyrimidinase used for the hydrolytic opening of non-natural 6-monosubstituted dihydrouracils, which may be exploited for the production of β-amino acids.     

Indicators of environmentally sound land use in the humid tropics: The potential roles of expert opinion,knowledge engineering and knowledge discovery

Despite abundant literature on indicators for sustainable resource management, practical tools to help local users to apply its general concepts at a local to regional level are scarce. This means that decisions over land evaluation and land use at a local level are often not based on the formal application of indicators or decision support systems for environmentally sound management but instead on the opinion of local expertise, for instance forest managers, cattle breeders, farmers and/or academics. This is particularly seen to be the case in the tropics where access to modern communication and information technologies is restricted.As the opinions of experts are often based on and influenced by personal experience, intuition, heuristics and bias, their evaluations and decision are often unclear to the non-expert working at a local level. In order to make their reasoning more comprehensible to the non-expert, the ecological condition of 176 plots in the tropical Southeast of Mexico were evaluated by experts on soil fertility, forest management, cattle breeding and agriculture. With the assistance of a knowledge engineer (one who converts expert knowledge and reasoning into a model), these expert opinions and reasoning were then translated into a formal computer model.As an alternative approach we applied a knowledge discovery technique, namely the induction of regression trees and automatically developed models using the expert evaluations as training data. Where knowledge engineering was tedious and time consuming, regression models could be rapidly generated. Moreover, the correspondence between regression trees and expert opinions was considerably higher than the correspondence between expert opinion and their own models. The regression trees used less explicative variables than the models generated by the experts. The minimisation of sampling effort due to variable space reduction means that the application of regression tree induction has a high potential for a rapid development of indicators for narrowly defined ecological assessments, needed for decision making on a local or regional scale.     

Phylogeny of magpie‐robins and shamas (Aves: Turdidae: Copsychus and Trichixos): implications for island biogeography in Southeast Asia

Aim Magpie‐robins and shamas are forest and woodland birds of south Asia. There are two genera: Trichixos for the monotypic T. pyrrhopygus, and Copsychus for other species. Two species are widespread, whereas the others are restricted to specific islands. Endemicity is highest in the Philippines. Using phylogenetic methods, we examined how this group came to its unusual distribution. Location Mainland Asia from India to southern China, and islands from Madagascar to the Philippines. Particular emphasis is placed on the Greater Sundas and Philippines. Methods The phylogeny was estimated from DNA sequences of 14 ingroup taxa representing all nine currently recognized Copsychus and Trichixos species. The entire mitochondrial ND2 gene and portions of nuclear myoglobin intron 2 (Myo2) and transforming growth factor beta 2 intron 5 (TGFβ2‐5) were sequenced for all but two species. The phylogeny was reconstructed using maximum likelihood and Bayesian methods. The timing of divergence events was estimated using a relaxed molecular clock approach, and ancestral areas were examined using stochastic modelling. Results The group comprises three main clades corresponding to ecological types: Trichixos, a primary‐forest specialist; Copsychus magpie‐robins, open‐woodland and coastal species; and Copsychus shamas, thick‐forest species. Trichixos appears to be sister to the magpie‐robins, rendering Copsychus polyphyletic. The dating of phylogenetic nodes was too ambiguous to provide substantial insight into specific geographical events responsible for divergence within the group. Some patterns are nevertheless clear. Copsychus shamas reached the Philippines, probably in two separate invasions, and split into endemic species. Copsychus malabaricus and C. saularis expanded widely in the Greater Sundas and mainland Southeast Asia without species‐level diversification. Main conclusions Magpie‐robins are excellent dispersers and have diversified into distinct species only on isolated oceanic islands. Trichixos, a poor disperser, is restricted to mature forests of the Malay Peninsula, Sumatra and Borneo. Copsychus shamas are intermediate in habitat preference and dispersal capabilities. Their endemism in the Philippines may be attributed to early colonization and specialization to interior forests. In the Greater Sundas, C. malabaricus and C. saularis populations split and came together on Borneo to form two separate subspecies (of each species), which now hybridize.     

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.     

The classical complement cascade mediates CNS synapse elimination

During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.     

Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage

The present study describes the effects of exposure of bovine sperm to mild and more intense ROS generating conditions. The membrane integrity of the incubated sperm was assessed and the incubated sperm were used for IVF after which the percentages of cleavage and blastocyst formation were determined for a period up to 9 days. The incubated sperm samples showed increased levels of molecular oxidation in the plasma membrane, the mitochondria, the cytosol and to a lesser extent in the sperm's DNA. The sperm membrane integrity as well as the first cleavage rates obtained with sperm from mild ROS generating conditions (100 microM H2O2) were not different from sperm incubated without pro-oxidants. However, exposure of sperm to more severe oxidative stress (500 mM H2O2 or a combination of 100 microM ascorbic acid, 20 microM FeSO4 and 500 microM H2O2) led to plasma membrane oxidation, reduced percentages of cleaved embryos and a reduction in the percentages of cleaved embryos that developed to the blastocyst stage. From these results, we conclude that the impact of oxidative stress to sperm becomes primarily manifest after the first cleavage of the formed zygote. Importantly, the level of lipid peroxidation in the sperm plasma membrane significantly correlates with blastocyst formation when the corresponding sperm is used for in vitro fertilization of oocytes.     

Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.