Optimal pollinator attraction strategies in Trollius ranunculoides Hemsl. (Ranunculaceae) at different altitudes: increased floral display or promotion of nectar output?

For alpine plant species, patterns of resource allocation to functional floral traits for pollinator attraction can be highly significant in adaptation to low pollinator abundance and consequent pollen limitation. Increased pollination can be achieved either through a larger floral display or production of more pollen rewards. In this study, variation in resource allocation to different components for pollinator attraction was studied along an altitudinal gradient in Trollius ranunculoides, an obligate self‐incompatible out‐crosser of the Qinghai–Tibet Plateau. We compared resource allocation to conspicuous yellow sepals (which mainly provide visual attraction) and degenerate petals (which provide the major nectar reward) between populations at four altitudes. Furthermore, we investigated the contribution of sepals and petals to pollinator attraction and female reproductive success in an experiment with sepal or petal removal at sites at different altitudes. At the level of single flowers, resource allocation increased to sepals but decreased to petals with increasing altitude. Consistent with these results, sepals contributed much more to visitation rate and seed set than petals, as confirmed in the sepal or petal removal experiment. Sepals and petals contributed to female reproductive success by ensuring visitation rate rather than visitation duration. To alleviate increasing pollen limitation with increasing altitude, resource allocation patterns of T. ranunculoides altered to favour development of sepals rather than petals. This strategy may improve pollination and reproductive success through visual attraction (sepal) rather than nectar reward (petal) over a gradient of decreasing pollinator abundance.     

Roles of the DYRK kinase Pom2 in cytokinesis, mitochondrial morphology, and sporulation in fission yeast

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast.     

Targeted delivery of mutant tolerant anti-coxsackievirus artificial microRNAs using folate conjugated bacteriophage Phi29 pRNA

Background

Myocarditis is the major heart disease in infants and young adults. It is very commonly caused by coxsackievirus B3 (CVB3) infection; however, no specific treatment or vaccine is available at present. RNA interference (RNAi)-based anti-viral therapy has shown potential to inhibit viral replication, but this strategy faces two major challenges; viral mutational escape from drug suppression and targeted delivery of the reagents to specific cell populations.

Methodology/Principal Findings

In this study, we designed artificial microRNAs (AmiRs) targeting the 3′untranslated region (3′UTR) of CVB3 genome with mismatches to the central region of their targeting sites. Antiviral evaluation showed that AmiR-1 and AmiR-2 reduced CVB3 (Kandolf and CG strains) replication approximately 100-fold in both HeLa cells and HL-1 cardiomyoctes. To achieve specific delivery, we linked AmiRs to the folate-conjugated bacterial phage packaging RNA (pRNA) and delivered the complexes into HeLa cells, a folate receptor positive cancer cells widely used as an in vitro model for CVB3 infection, via folate-mediated specific internalization. We found that our designed pRNA-AmiRs conjugates were tolerable to target mutations and have great potential to suppress viral mutational escape with little effect on triggering interferon induction.

Conclusion/Significance

This study provides important clues for designing AmiRs targeting the 3′UTR of viral genome. It also proves the feasibility of specific deliver of AmiRs using conjugated pRNA vehicles. These small AmiRs combined with pRNA-folate conjugates could form a promising system for antiviral drug development.     

Reducing AsA Leads to Leaf Lesion and Defence Response in Knock-Down of the AsA Biosynthetic Enzyme GDP-D-Mannose Pyrophosphorylase Gene in Tomato Plant

As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.     

Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%.     

耦合中空纤维膜超滤分离游离细胞催化合成ATP

对耦合中空纤维膜超滤分离进行游离细胞催化合成ATP过程进行了实验研究,考察了细胞的催化效率和膜组件的操作稳定性。结果显示,中空纤维超滤膜的耦合分离能有效地截留反应液中的游离酶,其中乙醇脱氢酶(ADH)和已糖激酶(HK)的稳态截留效率在95%以上。耦合膜分离的酵母细胞催化ATP合成反应可重复使用2.5～3.0次,酶的利用率比普通分离的细胞提高2.0～2.5倍。中空纤维超滤膜于0.1Mpa工作压力下连续11批耦合分离操作,膜的渗透性无明显下降,过滤速率保持在初速率的95%以上。在稀释速率0.25h-1下,反应体系保持了连续5h的ATP高转化率合成与分离耦合的拟稳态操作。     

Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447

Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k(cat) of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k(cat) of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k(cat) for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor beta (PDGFRbeta) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRbeta-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRbeta-dependent pathway tested.