Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR

Saturation transfer difference NMR experiments on human blood group B alpha-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and beta-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that beta-D-galactose and alpha-D-galactose bind weakly to GTB. Whereas beta-D-galactose binds to the acceptor and donor sites, it is suggested that alpha-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg(2+) and Mn(2+) is more relevant under physiological conditions.     

Evaluation of the relative amount of specific gaba-benzodiazepine receptor mrna after injection of total poly(A+)-rna into xenopus oocytes

GABA-benzodiazepine receptor-chloride channel complexes have been detected by electrophysiological recording in Xenopus oocytes previously injected with messenger RNA extracted either from optic lobes of chick embryos or from adult rat hippocampi. The ability of the oocyte to correctly translate exogenous messengers was used to develop a routine method which could allow a quantitative evaluation of specific mRNA coding for GABA-benzodiazepine receptor proteins following an injection of a fixed amount of total poly(A+)-RNA. The conditions of the validation of this method have been determined.     

Novel Neuroprotective Multicomponent Therapy for Amyotrophic Lateral Sclerosis Designed by Networked Systems

Amyotrophic Lateral Sclerosis is a fatal, progressive neurodegenerative disease characterized by loss of motor neuron function for which there is no effective treatment. One of the main difficulties in developing new therapies lies on the multiple events that contribute to motor neuron death in amyotrophic lateral sclerosis. Several pathological mechanisms have been identified as underlying events of the disease process, including excitotoxicity, mitochondrial dysfunction, oxidative stress, altered axonal transport, proteasome dysfunction, synaptic deficits, glial cell contribution, and disrupted clearance of misfolded proteins. Our approach in this study was based on a holistic vision of these mechanisms and the use of computational tools to identify polypharmacology for targeting multiple etiopathogenic pathways. By using a repositioning analysis based on systems biology approach (TPMS technology), we identified and validated the neuroprotective potential of two new drug combinations: Aliretinoin and Pranlukast, and Aliretinoin and Mefloquine. In addition, we estimated their molecular mechanisms of action in silico and validated some of these results in a well-established in vitro model of amyotrophic lateral sclerosis based on cultured spinal cord slices. The results verified that Aliretinoin and Pranlukast, and Aliretinoin and Mefloquine promote neuroprotection of motor neurons and reduce microgliosis.     

Biodiversity and chemotaxonomy of <Emphasis Type="Italic">Preussia</Emphasis> isolates from the Iberian Peninsula

This work documents 32 new Preussia isolates from the Iberian Peninsula, including endophytic and saprobic strains. The morphological study of the teleomorphs and anamorphs was combined with a molecular phylogenetic analysis based on sequences of the ribosomal rDNA gene cluster and chemotaxonomic studies based on liquid chromatography coupled to electrospray mass spectrometry. Sixteen natural compounds were identified. On the basis of combined analyses, 11 chemotypes are inferred.     

DNA-methyltransferase activities in Streptomyces antibioticus

Abstract To quantitate ammonia production by the intestinal flora, ammonia levels in arterial blood and the venous effluent of the small and large bowel of conventional, selectively decontaminated, germ-free and gnotobiotic rats were measured.
When the anaerobic flora was removed by decontamination a significant decrease in ammonia levels was observed in the effluent of both the small and large intestine. Decontamination of aerobic flora did not result in depression of ammonia production. Gnotobiotic rats colonised with an anaerobic flora or with a mixed aerobic and anaerobic flora, showed a slight increase in ammonia levels. No increase in ammonia production was observed when rats were colonised with aerobic flora. These results indicate that the Enterobacteriaceae were not responsible for ammonia generation.
The increase in ammonia levels after colonisation with anaerobic or mixed anaerobic/aerobic flora did not completely restore ammonia levels, despite reaching bacterial counts which were comparable to those in conventional rats. This may be explained by the limited number of species with which the rats were colonized. The finding that aerobic flora does not significantly contribute to ammonia production suggests that neomycin, known to be exclusively effective against aerobic flora, must have other effects to explain improvement of hepatic encephalopathy.     

Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains

Two industrial effluents, a pre-fermentation effluent and a post-fermentation effluent from a wheat starch production plant, were used as substrates for fuel ethanol production in anaerobic batch cultures using minimal nutritional amendment. The performances of three metabolically engineered xylose-utilizing Saccharomyces cerevisiae strains: TMB 3001 expressing XYL1, XYL2 and XKS1, redox metabolism modulated CPB.CR1 and glucose de-repressed CPB.CR2, as well as a reference strain CEN.PK 113-7D not fermenting xylose, were evaluated. For the recombinant strains a glucose consumption phase preceded the xylose consumption phase. In both effluents, biomass and ethanol production occurred predominantly during the glucose consumption phase, whereas xylitol and glycerol formation were predominant in the xylose consumption phase. Total specific ethanol productivities on glucose were 6-fold higher than on xylose in the pre-fermentation effluent and 15-fold higher than on xylose in the post-fermentation effluent. CPB.CR1 showed impaired growth compared to the two other xylose-utilizing strains, but displayed 18% increased ethanol yield in the post-fermentation effluent.     

Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2-10 tandem repeat sequence copies (mean=4.82 copies) while ALS6 alleles had 2-8 copies (mean=4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5' of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.     

Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.     

An experimental evaluation of a loop versus a reference design for two-channel microarrays

MOTIVATION: Despite theoretical arguments that so-called 'loop designs' for two-channel DNA microarray experiments are more efficient, biologists continue to use 'reference designs'. We describe two sets of microarray experiments with RNA from two different biological systems (TPA-stimulated mammalian cells and Streptomyces coelicolor). In each case, both a loop and a reference design were used with the same RNA preparations with the aim of studying their relative efficiency. RESULTS: The results of these experiments show that (1) the loop design attains a much higher precision than the reference design, (2) multiplicative spot effects are a large source of variability, and if they are not accounted for in the mathematical model, for example, by taking log-ratios or including spot effects, then the model will perform poorly. The first result is reinforced by a simulation study. Practical recommendations are given on how simple loop designs can be extended to more realistic experimental designs and how standard statistical methods allow the experimentalist to use and interpret the results from loop designs in practice. AVAILABILITY: The data and R code are available at http://exgen.ma.umist.ac.uk CONTACT: veronica.vinciotti@brunel.ac.uk.     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.