Gene Discovery and Molecular Dissection of Fructan Metabolism in Perennial Ryegrass (Lolium Perenne)

Fructans are the main soluble carbohydrate stored in up to a third of the vegetation of the earth, including the economically important temperate grasses. Fructans are polymers of fructose attached to a sucrose precursor. Perennial ryegrass (L. perenne L.) accumulates fructans of the inulin series, inulin neoseries and levan neoseries. Four enzymes are required to produce fructans of this profile: 1-SST (sucrose:sucrose 1- fructosyltransferase), 1-FFT (fructan:fructan 1-fructosyltransferase), 6G-FFT (6-glucose fructosyltransferase) and 6-FFT (fructan:fructan 6-fructosyltransferase) or 6-SFT (sucrose:fructan 6-fructosyltransferase) (Figure 1). Fructan biosynthetic enzymes have evolved from invertases and thus it is argued that fructan metabolism is an extension of sucrose metabolism. A high fructan content is a valuable resource in perennial ryegrass as it can be readily mobilised to sustain regrowth immediately after defoliation as well as adding to the nutritive value of the feed. However, the physiological role of fructans in grasses is not fully understood

An Intact Kidney Slice Model to Investigate Vasa Recta Properties and Function in situ

Background: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. Results: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow

Analysis of Expressed Sequence Tags of the Cyclically Parthenogenetic Rotifer Brachionus plicatilis

Background. Rotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking. Methodology/Principal Findings. We generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy. Conclusions/Significance. Despite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript-level data for B. plicatilis, and more generally of rotifers and other gnathiferan phyla, and can be browsed and searched at gmod.mbl.edu

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species

Vocation and Altruism in Nursing: The Habits of Practice

Background:At a time when British nursing has been under scrutiny for an apparent lack of compassion in education and practice, this paper based offers a perspective on the notions of vocation and altruism in nursing.Objectives:To understand the vocational and altruistic motivations of nurses through the application of Pierre Bourdieu's concepts of ‘symbolic capital’, ‘field’ and ‘habitus’ through a long interview with nurse respondents.Research design:A reflexive qualitative study was undertaken using the long interview. A thematic analysis of the data, using a qualitative data software package for analysis, was undertaken. The ideas of Pierre Bourdieu (Habitus, Capital and Field) were used to analyse and explain the content of community nurses' ‘talk’.Participants:Twelve Community Nurses working in a variety of local primary care settings volunteered to participate in the study. It was a self-selecting convenience sample of nurses responding to an invitation to be interviewed.Research context:A study in support of a doctoral thesis conducted within NHS primary care settings with registered nurses.Ethical considerations:The key considerations for this study were to be mindful of the possibility of emotional harm or distress being caused to the respondents during the retelling of their experiences. It was also essential to ensure that the locations or names of patients or staff (if discussed) were anonymised. Ethical approval was sought and granted by both the Local NHS Primary Care and the University Ethics committees before the study commenced.Findings:The nurse respondents had highly individual and at times contradictory views on their motivations to nurse including their views on vocation and altruism in nursing careers.Discussion:Bourdieu's ideas apply well to the nursing context and provided a useful theoretical framework to explore the social and cultural influences on nursing careers. Gender is important consideration in all aspects of nursing but class and educational experience is an important dimension in the stories nurses tell.Conclusion:The culturally determined experiences of these nurses in practice offer an authentic and realistic insight into the complex motives and predispositions of community nurses. This paper argues that nurse motivations whether vocational or altruistic, are better understood as culturally, rather than spiritually, driven