Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.     

Effect of fluid mechanical stresses and plasma constituents on aggregation of LDL

LDL aggregates when exposed to even moderate fluid mechanical stresses in the laboratory, yet its half-life in the circulation is 2-3 days, implying that little aggregation occurs. LDL may be protected from aggregation in vivo by components of plasma, or by a qualitative difference in flows. Previous studies have shown that HDL and albumin inhibit the aggregation induced by vortexing. Using a more reproducible method of inducing aggregation and assessing aggregation both spectrophotometrically and by sedimentation techniques, we showed that at physiological concentrations, albumin is the more effective inhibitor, and that aggregation is substantially but not completely inhibited in plasma. Heat denatured and fatty-acid-stripped albumin were more effective inhibitors than normal albumin, supporting the idea that hydrophobic interactions are involved. Aggregation of LDL in a model reproducing several aspects of flow in the circulation was 200-fold slower, but was still inhibited by HDL and albumin, suggesting similar mechanisms are involved. Within the sensitivity of our technique, LDL aggregation did not occur in plasma exposed to these flows. Thus, as a result of the characteristics of blood flow and the inhibitory effects of plasma components, particularly albumin, LDL aggregation is unlikely to occur within the circulation.     

An artificially designed pore-forming protein with anti-tumor effects

Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first native-like pore-forming protein, small globular protein (SGP). We show here that this artificially engineered protein has membrane-disrupting properties and anti-tumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications.     

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird

The use of stable isotopes to infer diet requires quantifying the relationship between diet and tissues and, in particular, knowing of how quickly isotopes turnover in different tissues and how isotopic concentrations of different food components change (discriminate) when incorporated into consumer tissues. We used feeding trials with wild-caught yellow-rumped warblers (Dendroica coronata) to determine delta15N and delta13C turnover rates for blood, delta15N and delta13C diet-tissue discrimination factors, and diet-tissue relationships for blood and feathers. After 3 weeks on a common diet, 36 warblers were assigned to one of four diets differing in the relative proportion of fruit and insects. Plasma half-life estimates ranged from 0.4 to 0.7 days for delta13C and from 0.5 to 1.7 days for delta15N . Half-life did not differ among diets. Whole blood half-life for delta13C ranged from 3.9 to 6.1 days. Yellow-rumped warbler tissues were enriched relative to diet by 1.7-3.6% for nitrogen isotopes and by -1.2 to 4.3% for carbon isotopes, depending on tissue and diet. Consistent with previous studies, feathers were the most enriched and whole blood and plasma were the least enriched or, in the case of carbon, slightly depleted relative to diet. In general, tissues were more enriched relative to diet for birds on diets with high percentages of insects. For all tissues, carbon and nitrogen isotope discrimination factors increased with carbon and nitrogen concentrations of diets. The isotopic signature of plasma increased linearly with the sum of the isotopic signature of the diet and the discrimination factor. Because the isotopic signature of tissues depends on both elemental concentration and isotopic signature of the diet, attempts to reconstruct diet from stable isotope signatures require use of mixing models that incorporate elemental concentration.     

Platinum carboxylato-pendant-arm macrocycles: structure, redox properties and anti-cancer potential

In an attempt to generate new platinum compounds that may be effective in the treatment of cancer, as well as having a lower toxicity than traditional platins and being orally viable, we are studing the synthesis and reactivity of platinum complexes of tetraazamacrocycles bearing carboxylato pendant arms. We have synthesized adducts of meso- and rac-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane-1,7-diacetic acid (L(1)H(2)). The meso-Pt(II)L(1) complex is unstable with respect to disproportionation, forming platinum metal and [meso-Pt(IV)L(1)](2+). The rac-isomer shows less tendency to disproportionate. Cyclic voltammetry suggests that the rac-Pt(II)L(1) complex undergoes two one-electron oxidations. Using bis-triazacyclononanenickel(III), [Ni(III)(tacn)(2)](3+) as an outer-sphere oxidant, the self-exchange rate for the [Pt(II/III)L](0/+) couple has been estimated at 0.034 M(-1) s(-1).     

N-losses and energy use in a scenario for conversion to organic farming

The aims of organic farming include the recycling of nutrients and organic matter and the minimisation of the environmental impact of agriculture. Reduced nitrogen (N)-losses and energy (E)-use are therefore fundamental objectives of conversion to organic farming. However, the case is not straightforward, and different scenarios for conversion to organic farming might lead to reduced or increased N-losses and E-use. This paper presents a scenario tool that uses a Geographical Information System in association with models for crop rotations, fertilisation practices, N-losses, and E-uses. The scenario tool has been developed within the multidisciplinary research project Land Use and Landscape Development Illustrated with Scenarios (ARLAS). A pilot scenario was carried out, where predicted changes in N-losses and E-uses following conversion to organic farming in areas with special interests in clean groundwater were compared. The N-surplus and E-use were on average reduced by 10 and 54%, respectively. However, these reductions following the predicted changes in crop rotations, livestock densities, and fertilisation practices were not large enough to ensure a statistically significant reduction at the 95% level. We therefore recommend further research in how conversion to organic farming or other changes in the agricultural practice might help to reduce N-surpluses and E-uses. In that context, the presented scenario tool would be useful.     

Formation of fibrin gel in fibrinogen-thrombin system: static and dynamic light scattering study

The dynamics of thrombin-induced fibrin gel formation was investigated by means of static and dynamic light scattering. The decay time distribution function, obtained by the dynamic light scattering, clearly revealed a stepwise gelation process: the formation of fibrin and protofibril from fibrinogen followed by the lateral aggregation of protofibrils to form fibrin fibers and the formation of a three-dimensional network consisting of fibers. This conversion process was correlated with the angular dependence of the scattered light intensity (static light scattering). The correlation function of dynamic light scattering was analyzed in terms of sol-gel transition and gel structure. The correlation function showed a stretched exponential type behavior before the sol to gel transition point, and it showed a power law behavior at the gelation point.     

Are hummingbirds facultatively ammonotelic? Nitrogen excretion and requirements as a function of body size

Most birds are uricotelic. An exception to this rule may be nectar-feeding birds, which excrete significant amounts of ammonia under certain conditions. Although ammonia is toxic, because it is highly water soluble its excretion may be facilitated in animals that ingest and excrete large amounts of water. Bird-pollinated plants secrete carbohydrate- and water-rich floral nectars that contain exceedingly little protein. Thus, nectar-feeding birds are faced with the dual challenge of meeting nitrogen requirements while disposing of large amounts of water. The peculiar diet of nectar-feeding birds suggests two hypotheses: (1) these birds must have low protein requirements, and (2) when they ingest large quantities of water their primary nitrogen excretion product may be ammonia. To test these hypotheses, we measured maintenance nitrogen requirements (MNR) and total endogenous nitrogen losses (TENL) in three hummingbird species (Archilochus alexandri, Eugenes fulgens, and Lampornis clemenciae) fed on diets with varying sugar, protein, and water content. We also quantified the form in which the by-products of nitrogen metabolism were excreted. The MNR and TENL of the hummingbirds examined were exceptionally low. However, no birds excreted more than 50% of nitrogen as ammonia or more nitrogen as ammonia than urates. Furthermore, ammonia excretion was not influenced by either water or protein intake. The smallest species (A. alexandri) excreted a significantly greater proportion (>25%) of their nitrogenous wastes as ammonia than the larger hummingbirds ( approximately 4%). Our results support the hypothesis that nectar-feeding birds have low protein requirements but cast doubt on the notion that they are facultatively ammonotelic. Our data also hint at a possible size-dependent dichotomy in hummingbirds, with higher ammonia excretion in smaller species. Differences in proportionate water loads and/or postrenal modification of urine may explain this dichotomy.     

Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases

Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6- centromeric exon 7- telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6- centromeric exon 7- telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.