The phenotypic expression of QTLs for partial resistance to barley leaf rust during plant development

Partial resistance is generally considered to be a durable form of resistance. In barley, Rphq2, Rphq3 and Rphq4 have been identified as consistent quantitative trait loci (QTLs) for partial resistance to the barley leaf rust pathogen Puccinia hordei. These QTLs have been incorporated separately into the susceptible L94 and the partially resistant Vada barley genetic backgrounds to obtain two sets of near isogenic lines (NILs). Previous studies have shown that these QTLs are not effective at conferring disease resistance in all stages of plant development. In the present study, the two sets of QTL–NILs and the two recurrent parents, L94 and Vada, were evaluated for resistance to P. hordei isolate 1.2.1 simultaneously under greenhouse conditions from the first leaf to the flag leaf stage. Effect of the QTLs on resistance was measured by development rate of the pathogen, expressed as latency period (LP). The data show that Rphq2 prolongs LP at the seedling stage (the first and second leaf stages) but has almost no effect on disease resistance in adult plants. Rphq4 showed no effect on LP until the third leaf stage, whereas Rphq3 is consistently effective at prolonging LP from the first leaf to the flag leaf. The changes in the effectiveness of Rphq2 and Rphq4 happen at the barley tillering stage (the third to fourth leaf stages). These results indicate that multiple disease evaluations of a single plant by repeated inoculations of the fourth leaf to the flag leaf should be conducted to precisely estimate the effect of Rphq4. The present study confirms and describes in detail the plant development-dependent effectiveness of partial resistance genes and, consequently, will enable a more precise evaluation of partial resistance regulation during barley development.     

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model

Introduction

The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA).

Methods

Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE₂ and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05.

Results

A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE₂ (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone.

Conclusion

Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.     

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.     

人微小病毒3个主要蛋白基因真核表达载体的构建

目的:分别克隆人细小病毒B19三个主要蛋白VP1、VP2、NS1全长基因,构建真核表达载体。方法:利用PCR和分子克隆技术,分别将B19病毒vp1、vp2、ns1基因全长片段扩增后,构建带荧光标签的真核表达载体;在人体细胞中表达并通过荧光、RT-PCR和Western Blot、测序等方法鉴定。结果:成功构建了包含B19病毒vp1、vp2、ns1全长基因,并在人体细胞中表达了VP1、VP2、NS1蛋白。结论:人微小病毒B19三个主要蛋白基因得到克隆和表达,为进行相关的研究奠定了基础。     

SUMMARY OF GREEN PLANT PHYLOGENY AND CLASSIFICATION

Abstract— A cladogram of green plants involving all major extant groups of green algae, bryophytes, pteridophytes, and seed plants is presented. It is partly based on contributions by B. Mishler and S. Churchill, H. Wagner, and P. Crane. The relationships of green plants to other green organisms ( Prochloron , euglenophytes) are discussed. The characters and subclades of the cladogram are briefly discussed, with an attempt to indicate weak points. The possibility of including some major extinct groups is considered. A cladistic classification consistent with the cladogram is presented. Grades are abandoned as taxa and major clades like the division Chlorophyta (green algae excluding micro-monadophytes and charophytes sensu Mattox and Stewart), the division Streptophyta (charophytes + embryophytes), the subdivision Embryophytina (land plants or embryophytes), the superclass Tracheidatae (tracheophytes), and the class Spermatopsida (seed plants) are recognized.