Carbon isotope ratios in ear parts of triticale : influence of grain filling

Four triticale (×Triticosecale Wittmack) genotypes were grown under rainfed conditions with limited irrigation support in Lleida in northeast Spain. For each variety, samples consisting of 10 tillers with half-sterilized spikes were taken three times from anthesis to maturity. Carbon isotope ratios (δ¹³C) were then determined in water extracts from ear bracts (glumes, paleas, and lemmas), awns and flag leaves, and in powdered kernels. For the half-sterilized spikes, carbon isotope analysis was carried out separately in bracts and awns from fertile and nonfertile spikelets. The δ¹³C in the water-soluble fraction of awns, glumes, and glumells from fruitless spikelets was significantly higher than that from fertile spikelets sampled at mid-grain filling. Differences in δ¹³C among sterile and fertile spikelets were not significant in samples taken a few days after anthesis or at maturity. These results are in accordance with some degree of refixation by awns and ear bracts of the CO₂ respired by grains during grain filling. There was progressively higher δ¹³C from flag leaf blades to awns, glumes, and glumells. This variation in δ¹³C along plant parts may be caused by differences in the ratio of assimilation rate to CO₂-diffusive conductance. Values of δ¹³C of mature kernels were between the values at anthesis and mid-grain filling for the water-soluble fraction of flag leaves and inner bracts and were fairly similar to those of glumes and awns.     

Mutation of regulatory serines of rat tyrosine hydroxylase to glutamate: effects on enzyme stability and activity

Tyrosine hydroxylase is phosphorylated at four serine residues in its amino-terminus by multiple kinases. Phosphorylation of serine 40 by cAMP-dependent protein kinase results in alleviation of dopamine inhibition [J. Biol. Chem. 267 (1992) 12639]. The other serines are at positions 8, 19, and 31. The effect of phosphorylation at these serines has been investigated using mutated forms of tyrosine hydroxylase containing glutamates at the positions of the serines. The S8E, S19E, and S31E tyrosine hydroxylase variants have similar steady-state kinetic parameters and similar binding affinity for catecholamines to wild-type enzyme. The S8E, S19E, S31E, and S40E variants differ in stability at elevated temperatures. The S40E variant is the least stable, while the others are all more stable than wild-type enzyme. The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation. It may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosine hydroxylase which have been phosphorylated.     

The origins of Iberian horses assessed via mitochondrial DNA

Despite a number of recent studies that have focused on the origin of domestic horses, genetic relationships between major geographical clusters still remain poorly understood. In this study we analyzed a 296 bp mtDNA fragment from the HVI region of 171 horses representing 11 native Iberian, Barb, and Exmoor breeds to assess the maternal phylogeography of Iberian horses. The mtDNA haplogroup with a CCG motif (nucleotide position 15,494 to 15,496) was the most frequent in Iberian and Barb breeds (0.42 and 0.57, respectively), regardless of geographic location or group of breeds. This finding supports the close genetic relationship between the ancestral mare populations of the Iberian Peninsula and Northern Africa. Phenotypic differences among the Northern and Southern Iberian groups of breeds are not explained by population subdivision based on maternal lineages. Our results also suggest that Northern Iberian ponies--which are phenotypically close to British ponies, especially Exmoor--are the result of an introgression rather than population replacement.     

A biotic index using the seagrass Posidonia oceanica (BiPo), to evaluate ecological status of coastal waters

The development of ecologically based indices that respond to disturbances in a predictable manner has been stressed by the EU Water Framework Directive. The seagrass Posidonia oceanica, given its ecological indicator characteristics, has been identified as one of the elements to determine ecological status under the EU Water Framework Directive. The purpose of this study is therefore to develop a biotic index based on P. oceanica (BiPo), focussing on: (i) the necessity of an index that may be applied over the largest geographical extent possible, (ii) the necessity of a tool for a baseline evaluation of P. oceanica status in the Mediterranean, (iii) the compliance with WFD requirements, (iv) the efficiency of the method in terms of reliability and cost. The BiPo index is developed on the basis of all P. oceanica monitoring data available in the western Mediterranean and on a standard assessment of anthropogenic pressures. The index metrics are selected and evaluated on the basis of this pressures assessment, and are subsequently integrated for the evaluation of ecological status. The index is then tested on 15 sites around Corsica (France). The results show that the BiPo well reflects meadow health status and ecological status. Furthermore it is reliable, standard and cost-effective, and can be applied to a wide array of management and conservation purposes.     

Kinetics of regulatory serine variants of tyrosine hydroxylase with cyclic AMP-dependent protein kinase and extracellular signal-regulated protein kinase 2

Rat tyrosine hydroxylase is phosphorylated at four serine residues, at positions 8, 19, 31, and 40 in its amino terminal regulatory domain by multiple protein kinases. Cyclic AMP-dependent protein kinase phosphorylates S40, which results in alleviation of inhibition by dopamine. Extracellular signal-regulated protein kinase 2 phosphorylates S8 and S31. Site-directed serine-to-glutamate mutations were introduced into tyrosine hydroxylase to mimic prior phosphorylation of the regulatory serines; these proteins were used as substrates for cAMP-dependent kinase and extracellular signal-regulated kinase 2. The activity of cAMP-dependent kinase was unaffected by the substitution of serines 8, 19 or 31 with glutamate and the activity of extracellular signal-regulated kinase 2 was unaffected by substitution of serines 19 or 40 with glutamate. Cyclic AMP-dependent kinase was less active in phosphorylating S40 if dopamine was bound to tyrosine hydroxylase, but extracellular signal-regulated kinase 2 phosphorylation at S31 was unaffected by the presence of dopamine.     

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1

The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5β1 and αVβ3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL-αVβ3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVβ3. Furthermore, structure-activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVβ3 are proposed.     

Engineered Salmonella allows real-time heterologous gene expression monitoring within infected zebrafish embryos

Microbial host-pathogen interactions have been traditionally well studied at genetic and physiological levels, but cell-resolution analyses have been particularly scarce. This has been especially remarkable for intracellular parasites for two major reasons: first, the inherent loss of bacteria traceability once infects its hosts; second and more important, the limited availability of genetic tools that allow a tight regulated expression of bacterial virulence genes once inside the host tissues. Here we present novel data supporting the use of zebrafish embryos to monitor Salmonella enterica serovar Thyphimurium infection. Intravenous infection of Salmonella can be easily monitored using in vivo fluorescence that allows the visualization of free-swimming bacteria through the circulatory system. Moreover, we have engineered Salmonella to voluntarily activate heterologous gene expression at any point during infection once inside the zebrafish macrophages using a salicylate-based expression system. This approach allows real-time cell-resolution in vivo monitoring of the infection. All together, this approach paves the road to cell-based resolution experiments that would be harder to mimic in other vertebrate infection models.