Effect of alpha-lipoic acid on vascular responses and nociception in diabetic rats.

Oxidative stress contributes to the vascular and neurological complications of diabetes mellitus. The aim was to evaluate the effects of treatment with the radical scavenger and transition metal chelator, alpha-lipoic acid, on endothelium-dependent relaxation of the mesenteric vasculature and on superior cervical ganglion blood flow in 8 week streptozotocin-induced diabetic rats. alpha-Lipoic acid effects on small nerve fiber-mediated nociception were also monitored. For the in vitro phenylephrine-precontracted mesenteric vascular bed, diabetes caused a 31% deficit in maximum endothelium-dependent relaxation to acetylcholine, and a 4-fold reduction in sensitivity. alpha-Lipoic acid gave 85% protection against these defects. Acetylcholine responses are mediated by nitric oxide and endothelium-derived hyperpolarizing factor: isolation of the latter by nitric oxide synthase blockade revealed a 74% diabetic deficit that was halved by alpha-lipoic acid. Superior cervical ganglion blood flow, 52% reduced by diabetes, was dose-dependently restored by alpha-lipoic acid (ED(50), 44 mg/kg/d). Diabetic rats exhibited mechanical and thermal hyperalgesia, which were abolished by alpha-lipoic acid treatment. Thus, diabetes impairs nitric oxide and endothelium-derived hyperpolarizing factor-mediated vasodilation. This contributes to reduced neural perfusion, and may be responsible for altered nociceptive function. The effect of alpha-lipoic acid strongly implicates oxidative stress in these events and suggests a potential therapeutic approach.     

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

Background

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.

Methodology

Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.

Conclusions

The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.     

Pyruvate dehydrogenase phosphatase 1 (<Emphasis Type="Italic">PDP1</Emphasis>) null mutation produces a lethal infantile phenotype

Pyruvate dehydrogenase phosphatase deficiency has previously only been confirmed at the molecular level in two brothers and two breeds of dog with exercise intolerance. A female patient, who died at 6 months, presented with lactic acidemia in the neonatal period with serum lactate levels ranging from 2.5 to 17 mM. Failure of dichloroacetate to activate the PDH complex in skin fibroblasts was evident, but not in early passages. A homozygous c.277G > T (p.E93X) nonsense mutation in the PDP1 gene was identified in genomic DNA and immunoblotting showed a complete absence of PDP1 protein in mitochondria. Native PDHC activity could be restored by the addition of either recombinant PDP1 or PDP2. This highlights the role of PDP2, the second phosphatase isoform, in PDP1-deficient patients for the first time. We conclude that the severity of the clinical course associated with PDP1 deficiency can be quite variable depending on the exact nature of the molecular defect.     

Mycorrhizal suppression and phosphorus addition influence the stability of plant community composition and function in a temperate steppe

Nutrient enrichment can reduce ecosystem stability, typically measured as temporal stability of a single function, e.g. plant productivity. Moreover, nutrient enrichment can alter plant–soil interactions (e.g. mycorrhizal symbiosis) that determine plant community composition and productivity. Thus, it is likely that nutrient enrichment and interactions between plants and their soil communities co-determine the stability in plant community composition and productivity. Yet our understanding as to how nutrient enrichment affects multiple facets of ecosystem stability, such as functional and compositional stability, and the role of above–belowground interactions are still lacking. We tested how mycorrhizal suppression and phosphorus (P) addition influenced multiple facets of ecosystem stability in a three-year field study in a temperate steppe. Here we focused on the functional and compositional stability of plant community; functional stability is the temporal community variance in primary productivity; compositional stability is represented by compositional resistance, turnover, species extinction and invasion. Community variance was partitioned into population variance defined as community productivity weighted average of the species temporal variance in performance, and species synchrony defined as the degree of temporal positive covariation among species. Compared to treatments with mycorrhizal suppression, the intact AM fungal communities reduced community variance in primary productivity by reducing species synchrony at high levels of P addition. Species synchrony and population variance were linearly associated with community variance with the intact AM fungal communities, while these relationships were decoupled or weakened by mycorrhizal suppression. The intact AM fungal communities promoted the compositional resistance of plant communities by reducing compositional turnover, but this effect was suppressed by P addition. P addition increased the number of species extinctions and thus promoted compositional turnover. Our study shows P addition and AM fungal communities can jointly and independently modify the various components of ecosystem stability in terms of plant community productivity and composition.     

Measures of human olfactory perception during pregnancy

Although considerable anecdotal evidence suggests that pregnancy affects olfactory sensitivity, scientific evidence is limited and inconclusive. Whereas hedonic ratings are affected by pregnancy, odor identification is not. The aim of the current study was to examine odor perception in women across pregnancy and in the postpartum period. One hundred nonsmoking women who were pregnant, postpartum, or had never been pregnant were tested on the University of Pennsylvania Smell Identification Test (UPSIT). Intensity ratings and scratch patterns were collected as potential indicators of sensitivity, and participants rated the odors' pleasantness. Participants also rated their own sense of smell. Mean UPSIT scores did not differ significantly across groups indicating no difference in odor identification. Trends in planned comparisons suggested that in the first trimester, odors were rated as more intense and less pleasant. In the first trimester, women scratched the odor strips significantly fewer times. Consistent with previous reports, 90% of pregnant women reported that specific odors smelled less pleasant and 60% reported that some odors smelled more pleasant. Although nearly two-thirds of pregnant women rated their olfactory sensitivity to be enhanced during pregnancy and overall pregnant women's self-rated olfactory sensitivity was higher than controls', self-ratings were not correlated with UPSIT scores nor odor intensity ratings. These results suggest that these and previous findings may reflect the fact that the effect of pregnancy on olfaction is small and inconsistent.     

The potential for large carnivores to act as biodiversity surrogates in southern Africa

Biodiversity in southern Africa is globally extraordinary but threatened by human activities. Although there are considerable biodiversity conservation initiatives within the region, no one has yet assessed the potential use of large carnivores in such actions. Surrogate approaches have often been suggested as one such way of capitalizing on large carnivores. Here we review the suitability of the large carnivore guild (i.e., brown hyaena Hyaena hyaena, spotted hyaena Crocuta crocutta, cheetah Acinonyx jubatus, leopard Panthera pardus, lion Panthea leo and African wild dog Lycaon pictus) to act as surrogate species for biodiversity conservation in southern Africa. We suggest that the guild must be complete for the large carnivores to fully provide their role as ecological keystones. The potential for large carnivores to act as umbrella and indicator species seems limited. However, self-sustaining populations of large carnivores may be useful indicators of unfragmented landscapes. Moreover, diversity within the large carnivore guild may reflect overall biodiversity. Although the global appeal of the large African carnivores makes them important international flagships, we stress that international conservation funding must be linked to local communities for them to be important also locally. In summary, we suggest that the flagship value of these large carnivores should be used to promote biodiversity conservation in the region, and that the suggested relationship between large carnivore diversity and overall biodiversity is empirically tested. Finally we suggest that direct conservation activities should focus on enhancing the keystone values of large carnivores through complete guild conservation and restoration.     

Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit

The ChillPeach database was developed to facilitate identification of genes controlling chilling injury (CI), a global-scale post-harvest physiological disorder in peach. It contained 7,862 high-quality ESTs (comprising 4,468 unigenes) obtained from mesocarp tissues of two full-sib progeny contrasting for CI, about 48 and 13% of which are unique to Prunus and Arabidopsis, respectively. All ESTs are in the Gateway vector to facilitate functional assessment of the genes. The data set contained several putative SNPs and 184 unigenes with high quality SSRs, of which 42% were novel to Prunus. Microarray slides containing 4,261 ChillPeach unigenes were printed and used in a pilot experiment to identify differentially expressed genes in cold-treated compared to control mesocarp tissues, and in vegetative compared to mesocarp tissues. Quantitative RT-PCR (qRT-PCR) confirmed microarray results for all 13 genes tested. The microarray and qRT-PCR analyses indicated that ChillPeach is rich in putative fruit-specific and novel cold-induced genes. A website ( http://bioinfo.ibmcp.upv.es/genomics/ChillPeachDB ) was created holding detailed information on the ChillPeach database.