Fine mapping of the chromosome 5B region carrying closely linked rust resistance genes <Emphasis Type="Italic">Yr47</Emphasis> and <Emphasis Type="Italic">Lr52</Emphasis> in wheat

Key message

Fine mapping of Yr47 and Lr52 in chromosome arm 5BS of wheat identified close linkage of the marker sun180 to both genes and its robustness for marker-assisted selection was demonstrated.

Abstract

The widely effective and genetically linked rust resistance genes Yr47 and Lr52 have previously been mapped in the short arm of chromosome 5B in two F₃ populations (Aus28183/Aus27229 and Aus28187/Aus27229). The Aus28183/Aus27229 F₃ population was advanced to generate an F₆ recombinant inbred line (RIL) population to identify markers closely linked with Yr47 and Lr52. Diverse genomic resources including flow-sorted chromosome survey sequence contigs representing the orthologous region in Brachypodium distachyon, the physical map of chromosome arm 5BS, expressed sequence tags (ESTs) located in the 5BS6-0.81-1.00 deletion bin and resistance gene analog contigs of chromosome arm 5BS were used to develop markers to saturate the target region. Selective genotyping was also performed using the iSelect 90 K Infinium wheat SNP assay. A set of SSR, STS, gene-based and SNP markers were developed and genotyped on the Aus28183/Aus27229 RIL population. Yr47 and Lr52 are genetically distinct genes that mapped 0.4 cM apart in the RIL population. The SSR marker sun180 co-segregated with Lr52 and mapped 0.4 cM distal to Yr47. In a high resolution mapping population of 600 F₂ genotypes Yr47 and Lr52 mapped 0.2 cM apart and marker sun180 was placed 0.4 cM distal to Lr52. The amplification of a different sun180 amplicon (195 bp) than that linked with Yr47 and Lr52 (200 bp) in 204 diverse wheat genotypes demonstrated its robustness for marker-assisted selection of these genes.

     

The effect of nitrogen on the movement of tracers down the stolon of Saxifraga sarmentosa,with some observations on the influence of light

Summary The movement of applied ¹³⁷Cs and of naturally-assimilated ¹⁴C down the long stolon of Saxifraga is strongly inhibited by confining a length of 10 to 30 cm of the stolon in an atmosphere of nitrogen. The inhibition is reversible, normal transport being restored after less then 4 h when the stolon is returned to air from 5 h in nitrogen. Callose formation does not seem to be involved. There is evidence that local darkness has a similar adverse effect on phloem transport.These findings are considered antagonistic to the pressure-flow hypothesis, but favourable to the active mass-flow theories.This work formed part of that submitted for the degree of Ph.D. of the University of London.     

Patterns of alien plant diversity in the urban landscapes of global biodiversity hotspots: a case study from the Himalayas

In an age of Anthropocene, the urban landscapes are recognised as the ‘hotspots’ of human-mediated alien species introductions. As the cities provide an ideal natural experimental system to investigate the patterns of alien plant diversity in urban landscapes, the present study aimed to unravel the taxonomic, biogeographic and ecological patterns of alien flora of Srinagar—one of the largest urban centres in the Himalayan biodiversity hotspot. The alien flora of Srinagar comprises 325 species, constituting ca.35% of total flora of the city. Out of the 325 alien species documented, 157 species (43%) were recorded to be under cultivation, while 168 species (57%) were growing in the wild (i.e., outside cultivation); those growing in the wild, in turn, comprised 110 cultivation escapes and 58 accidentally introduced plant species. Biogeographically, two-third of the alien plant diversity reported from Srinagar is native to Asia-Temperate. This indicates that climatic similarity between Asia-Temperate and Kashmir Himalayas facilitate in flourishing similar floristic diversity. The study highlights a relatively higher proportion of herbaceous growth form in the aliens growing in the wild (80%) than those under cultivation (43%). Similarly, 82% of the alien species under cultivation had a perennial life span, but those growing in the wild were dominated by annuals (44%). Currently, 45 species are growing as casuals and 124 species are naturalised (including 105 naturalised non-invasive and 19 naturalised invasive). Along the continuum of casual-naturalised-invasive categories, the contribution of cultivation escapes and accidently introduced aliens contrastingly shows decreasing and increasing trends respectively. Interestingly, the results revealed that the human practice of stopping cultivation of alien escapes increased rapidly as we move along the continuum. Thus, the present study has investigated the patterns of alien plant diversity in the urban landscape of Srinagar, and the results obtained offer scientific insights toward better scientific understanding and management of plant invasions in this Himalayan city, with wider policy implications for neighbouring urbanised landscapes in the Himalayas and other mountainous regions across the world.     

Infection with Host-Range Mutant Adenovirus 5 Suppresses Innate Immunity and Induces Systemic CD4+ T Cell Activation in Rhesus Macaques

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.     

Factors influencing the distribution of leopard in a semiarid landscape of Western India

Previously, factors governing distribution of leopard (Panthera pardus fusca) in forest habitats of the Indian subcontinent were unknown. The present study assessed the influence of different ecogeographic variables determining the distribution of leopards in and around Sariska Tiger Reserve through MaxEnt habitat suitability model based on camera trapping method. Camera trapping was used to collect presence/absence information in the study area from December 2008 to June 2010. Information of 11 macrohabitat characteristics and variables (habitat types, prey species, Normalized Difference Vegetation Index (NDVI), elevation, livestock, village, water source, etc.) were collected along with leopard presence data. The probability of presence of leopards increased with decreasing distance to water and increasing encounter rate of peafowl (Pavo cristatus), chital (Axis axis), sambar (Rusa unicolor), and wild pig (Sus scrofa). It was found that the probability of presence of leopards increased with increasing area of Zizyphus mixed forest patches and NDVI. Results of this study showed that the probability of presence of leopards was higher in habitat types with intermediate cover, high wild prey base, and water sources. They also indicated that leopards are not always ‘generalists’ showing some degree of specialization, at least in their choice of habitat, and this information is useful for conserving leopard in human-dominated landscapes.     

Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

l-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of l-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of l-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of l-[(3)H]carnitine uptake were investigated with or without specific inhibitors. l-Carnitine uptake in mature cells was sodium dependent and linear with time. K(m) and V(max) values for saturable uptake were 14.07 +/- 1.70 micro M and 26.3 +/- 0.80 pmol. mg protein(-1). 6 min(-1), respectively. l-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that l-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.     

Development and characterization of a series of soluble tetrameric and monomeric streptavidin muteins with differential biotin binding affinities

The strong biotin-streptavidin interaction limits the application of streptavidin as a reversible affinity matrix for purification of biotinylated biomolecules. To address this concern, a series of single, double, and triple streptavidin muteins with different affinities to biotin were designed. The strategy involves mutating one to three strategically positioned residues (Ser-45, Thr-90, and Asp-128) that interact with biotin and other framework structure-maintaining residues of streptavidin. The muteins were produced in soluble forms via secretion from Bacillus subtilis. The impact of individual residues on the overall structure of streptavidin is reflected by the formation of monomeric streptavidin to different extents. Of the three targeted residues, Asp-128 has the most dramatic effect (Asp-128 > Thr-90 > Ser-45). Conversion of all three targeted residues to alanine results in a soluble biotin binding mutein that exists 100% in the monomeric state. Both wild-type and mutated (monomeric and tetrameric) streptavidin proteins were purified, and their kinetic parameters (on- and off-rates) were determined using a BIAcore biosensor with biotin-conjugated bovine serum albumin immobilized to the sensor chip. This series of muteins shows a wide spectrum of affinity toward biotin (K(d) from 10(-6) to 10(-11) m). Some of them have the potential to serve as reversible biotin binding agents.     

Transforming growth factor Beta1 induction of tissue inhibitor of metalloproteinases 3 in articular chondrocytes is mediated by reactive oxygen species

Transforming growth factor beta1 (TGF-beta1) stimulates cartilage extracellular matrix synthesis but, in excess, evokes synovial inflammation, hyperplasia, and osteophyte formation in arthritic joints. TGF-beta1 induces tissue inhibitor of metalloproteinases 3 (TIMP-3), an inhibitor of cartilage-damaging matrix metalloproteianases and aggrecanases. We investigated the role of reactive oxygen species (ROS) in TIMP-3 induction by TGF-beta1. In primary human and bovine chondrocytes, ROS scavenger and antioxidant N-acetylcysteine (NAC) inhibited TGF-beta1-induced TIMP-3 mRNA and protein increases. Ebselen and ascorbate also reduced this induction. TGF-beta1 time-dependently induced ROS production that was suppressed by NAC. Hydrogen peroxide, a ROS, induced TIMP-3 RNA. The TIMP-3 increase induced by TGF-beta1 was partly Smad2-dependent. TGF-beta1-stimulated Smad2 phosphorylation was inhibited by NAC. Reduced glutathione and L-cysteine also blocked Smad2 and TIMP-3 induction by TGF-beta1, whereas a nonthiol, N-acetylalanine, did not. Smad2 was not activated by H2O2. Smad2 phosphorylation was independent, and TIMP-3 expression was dependent, on new protein synthesis. TGF-beta-stimulated ERK and JNK phosphorylation was also inhibited by NAC. However, inhibitory actions of NAC were not mediated by ERK activation. Thus, ROS mediate TGF-beta1-induced TIMP-3 gene expression. Blocking TGF-beta1-induced gene expression by modulating cellular redox status with thiols can be potentially beneficial for treating arthritic and other disorders caused by excessive TGF-beta1.     

L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2

Maternofetal transport of L-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that L-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K_m = 11.09 ± 1.32 µM; V_max = 41.75 ± 0.94 pmol·mg protein^–1·min^–1), and was unchanged over the pH range from 5.5 to 8.5. L-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of L-carnitine, including D-carnitine, acetyl-D,L-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-L-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na⁺-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for L-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans. membrane transport; valproate; maternofetal; xenobiotics; acylcarnitine     

14-3-3 connects glycogen synthase kinase-3 beta to tau within a brain microtubule-associated tau phosphorylation complex

In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3beta and tau are parts of an approximately 400-500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 zeta co-elutes with the tau phosphorylation complex components tau and GSK3 beta. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3 zeta, GSK3 beta, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3 beta, 14-3-3 zeta co-precipitates with GSK3 beta, indicating that GSK3 beta binds to 14-3-3 zeta. From HEK-293 cells transfected with tau, GSK3 beta, and 14-3-3 zeta in different combinations, tau co-immunoprecipitates with GSK3 beta only in the presence of 14-3-3 zeta. In vitro, approximately 10-fold more tau binds to GSK3 beta in the presence of than in the absence of 14-3-3 zeta. In transfected HEK-293 cells, 14-3-3 zeta stimulates GSK3 beta-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 zeta dimer simultaneously binds and bridges tau and GSK3 beta and stimulates GSK3 beta-catalyzed tau phosphorylation.