Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

gacS基因插入失活对绿针假单胞菌G-05的两种胞外次生代谢物合成的影响

一株来自大棚温室甜椒根际的绿针假单胞菌Pseudomonas chlororaphis G-05,可分泌抗生物质吩嗪-1-羧酸,并具有抑制辣椒疫霉的生物防治功效。【目的】为了系统研究该菌株的生物防治功能及抗生物质合成与分泌机制。【方法】首先通过生化法和16S rDNA同源比对法对该菌株进行系统分类的初步鉴定,再根据基因的同源性从G-05基因组DNA中克隆长1.4 kb的gacS基因的部分保守区段,采用抗庆大霉素基因（gentamycin resistance cassette, aacC1）插入失活的策略构建了该基因突变株G-05S。【结果】在King’ s B（KMB）或PPM培养基中,突变株G-05S合成吩嗪-1-羧酸的能力受到明显抑制。然而,突变株G-05S分泌的吲哚乙酸与野生株相比无显著差异。互补实验表明,gacS基因的表达可以使突变株G-05S的吩嗪-1-羧酸的合成恢复到野生株水平。【结论】由此推测,GacS(Global activator sensor )对不同次生代谢物的调控具有特异性。     

Understanding thermostability in cytochrome P450 by combinatorial mutagenesis

The cytochromes P450 are an important class of mono-oxygenases involved in xenobiotic metabolism and steroid biosynthesis in a diverse set of life forms. Discovery of CYP-119, a P450 from the archea Sulfolobus solfataricus has provided a means for understanding nature's method of stabilizing this important protein superfamily. To identify classes of stabilizing interactions used by CYP-119, we have generated a randomized library of point mutants and screened for mutants that are less thermostable than the wild type by monitoring the characteristic Soret band in the visible region of the cell lysis. The selected mutants were characterized by differential scanning calorimetry to compare the temperatures of the melting transitions of the various mutants. The identified mutations suggested that electrostatic interactions involving salt links and charge-charge interactions, as well as contributions from other interactions such as aromatic stacking, and side chain volume of hydrophobic residues contribute to enhanced thermostability in this cytochrome P450.     

中国尾孢菌属及其近似属的研究VIII.

报道钉孢属一个新种生在梾木属Cornussp.上的梾木钉孢Passaloracorni和1个新组合生在四叶重楼Parisquadrifolia.上的重楼钉孢Passaloraparidis;尚有尾孢菌属的4个中国新记录种南蛇藤生尾孢Cercosporacelastricola.,危地马拉尾孢Cercosporaguatemalensis,大薸尾孢Cercosporapistiae,和鼠尾草生尾孢Cercosporasalviicola。为新种提供了拉丁文简介、英文描述并绘图。标本保存在中国科学院微生物研究所菌物标本馆(HMAS)。     

Light intensity regulates growth and reproduction of a snail grazer (Gyraulus chinensis) through changes in the quality and biomass of stream periphyton

1. The light : nutrient hypothesis (LNH) proposes that herbivore growth rates are maximised at intermediate light‐to‐nutrient ratios. A reduction to light intensity (i.e. decreased light‐to‐nutrient ratio) should lead to reduced food availability for herbivores while excessive light intensity in oligotrophic environments (i.e. increased light‐to‐nutrient ratios) should increase the C : N and C : P ratios of producers. However, this hypothesis has not yet been supported by studies on stream ecosystems. 2. We tested the LNH by experimental application of controlled natural gradients in light intensity to oligotrophic laboratory channels that included periphyton and the freshwater snail Gyraulus chinensis. 3. The results in this oligotrophic environment indicate that light regulated the flow of matter between trophic levels and grazer reproduction by controlling C : P ratios of the producers.     

On phylogenetic tests of irreversible evolution

"Dollo's law" states that, following loss, a complex trait cannot reevolve in an identical manner. Although the law has previously fallen into disrepute, it has only recently been challenged with statistical phylogenetic methods. We employ simulation studies of an irreversible binary character to show that rejections of Dollo's law based on likelihood-ratio tests of transition rate constraints or on reconstructions of ancestral states are frequently incorrect. We identify two major causes of errors: incorrect assignment of root state frequencies, and neglect of the effect of the character state on rates of speciation and extinction. Our findings do not necessarily overturn the conclusions of phylogenetic studies claiming reversals, but we demonstrate devastating flaws in the methods that are the foundation of all such studies. Furthermore, we show that false rejections of Dollo's law can be reduced by the use of appropriate existing models and model selection procedures. More powerful tests of irreversibility require data beyond phylogenies and character states of extant taxa, and we highlight empirical work that incorporates additional information.     

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond—the flavinylation reaction—has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl-flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.     

Leaf nutrient concentration as an indicator of Populus and Tamarix response to flooding

Plants growing in infertile environments are able to produce more biomass per unit of nutrient taken up than plants of fertile habitats, and also to minimize nutrients loss by resorbing them from senescing leaves. The leaf nutrient concentration variability of two co-existing riparian tree genera (Populus and Tamarix) along a flood inundation gradient was examined to infer nutrient limitation and to compare nutrient use strategies in the two genera. To that end, seasonal and spatial variability in leaf nitrogen (N) and phosphorus (P) concentration (i.e., % dry mass of N and P) were analyzed in 720 samples of leaves (2 tree genera × 3 seasons × 12 sites × 10 tree replicates). Both Populus and Tamarix showed strong seasonal variability in leaf N and P concentrations, with values decreasing throughout the growing season. However, while N:P atomic ratio remained seasonally constant in Populus (N:P = 33), Tamarix shifted from N:P = 29 in spring to N:P = 36 and 37 in summer and fall. %N, %P and N:P atomic ratios were also spatially variable, but leaf litter N and P concentration (i.e., nutrient resorption proficiency) and leaf litter N:P generally followed the local flood inundation gradient as shown by linear mixed effects models. In particular, nutrient resorption was usually less proficient (higher terminal nutrient concentrations) at higher flood durations (in gravel bars and natural levees), whereas N:P increased in the drier sites (floodplain terrace). At floodplain level, a P-limitation that is higher than N-limitation seems to characterize the plant nutrient circulation in the riparian ecosystem studied. Tamarix was slightly more proficient in P resorption than Populus. The study shows that leaf nutrient concentration (e.g., N and P) derived from nutrient availability is partly controlled by the flood inundation regime and can be used as an indicator of nutrient limitation in forested floodplains. Subtle differences between tree genera provide an additional, novel explanation for the recent expansion of Tamarix in many arid and semi-arid rivers with altered hydrogeomorphic regimes.     

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97^UFD1/NPL4), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.     

Increased p wave dispersion in elite athletes

Background

Few studies have been performed on P wave indices in athletes. The aim of this study was to determine the behaviour of maximum P wave duration (Pmax), minimum P wave duration (Pmin) and P wave dispersion (PWD) in young high performance athletes, as well as the relationship of PWD with training history, heart rate (HR) and echocardiographic parameters.

Methods

We performed a cross-sectional observational study in 38 athletes of high performance in sports: water polo, distance running and weight lifting compared with 34 sedentary controls.

Results

The average age in both groups was 20.6 years. Note that PWD was increased in athletes (57 ± 14 ms vs. 40 ± 12 ms, p <0.001) while Pmin was significantly lower (57 ± 13 ms vs. 72 ± 13 ms, p <0.001), and there was no difference when comparing Pmax (114 ± 9 ms vs. 117 ± 14 ms, p> 0.05). The correlation between the duration of training (r = 0.511) and resting HR (r = 0.461) with PWD was significant (p <0.01).

Conclusions

PWD is increased in young athletes of high performance and was positively correlated with duration of training and baseline HR. The increase in PWD was secondary to a significant decrease in Pmin.