利用交换接头技术构建大麦条纹花叶病毒新疆株RNA_2的全长cDNA克隆

本文采用双股交换接头(Crossover linker)技术对已建立的大麦条纹花叶病毒新疆株(BSMV-XJ)RNA_2 cDNA重组质粒pUBS_(112)进行修饰。使cDNA两端不必要的poly(dG):poly(dC)尾准确地缺失,同时补上了cDNA中相对于RNA_2 5’端区缺少的三个核苷酸TAA,并在cDNA末端插入了预定的限制酶切顺序。通过原位杂交筛选、酶切图谱分析、cDNA两端序列测定等手段,证明已获得BSMV-XJ RNA_2组分的全长cDNA克隆。     

Ectopically expressed sweet pepper ferredoxin PFLP enhances disease resistance to Pectobacterium carotovorum subsp. carotovorum affected by harpin and protease‐mediated hypersensitive response in Arabidopsis

Plant ferredoxin‐like protein (PFLP) is a photosynthesis‐type ferredoxin (Fd) found in sweet pepper. It contains an iron–sulphur cluster that receives and delivers electrons between enzymes involved in many fundamental metabolic processes. It has been demonstrated that transgenic plants overexpressing PFLP show a high resistance to many bacterial pathogens, although the mechanism remains unclear. In this investigation, the PFLP gene was transferred into Arabidopsis and its defective derivatives, such as npr1 (nonexpresser of pathogenesis‐related gene 1) and eds1 (enhanced disease susceptibility 1) mutants and NAHG‐transgenic plants. These transgenic plants were then infected with the soft‐rot bacterial pathogen Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora ssp. carotovora, ECC) to investigate the mechanism behind PFLP‐mediated resistance. The results revealed that, instead of showing soft‐rot symptoms, ECC activated hypersensitive response (HR)‐associated events, such as the accumulation of hydrogen peroxide (H₂O₂), electrical conductivity leakage and expression of the HR marker genes (ATHSR2 and ATHSR3) in PFLP‐transgenic Arabidopsis. This PFLP‐mediated resistance could be abolished by inhibitors, such as diphenylene iodonium (DPI), 1‐l ‐trans‐epoxysuccinyl‐leucylamido‐(4‐guanidino)‐butane (E64) and benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐fmk), but not by myriocin and fumonisin. The PFLP‐transgenic plants were resistant to ECC, but not to its harpin mutant strain ECCAC5082. In the npr1 mutant and NAHG‐transgenic Arabidopsis, but not in the eds1 mutant, overexpression of the PFLP gene increased resistance to ECC. Based on these results, we suggest that transgenic Arabidopsis contains high levels of ectopic PFLP; this may lead to the recognition of the harpin and to the activation of the HR and other resistance mechanisms, and is dependent on the protease‐mediated pathway.     

FtsH-mediated repair of the photosystem II complex in response to light stress

A common feature of light stress in plants, algae, and cyanobacteria is the light-induced damage to the photosystem II complex (PSII), which catalyses the photosynthetic oxidation of water to molecular oxygen. A repair cycle operates to replace damaged subunits within PSII, in particular, the D1 reaction centre polypeptide, by newly synthesized copies. As yet the molecular details of this physiologically important process remain obscure. A key aspect of the process that has attracted much attention is the identity of the protease or proteases involved in D1 degradation. The results are summarized here of recent mutagenesis experiments that were designed to assess the functional importance of the DegP/HtrA and FtsH protease families in the cyanobacterium Synechocystis sp. PCC 6803. Based on these results and the analysis of Arabidopsis mutants, a general model for PSII repair is suggested in which FtsH complexes alone are able to degrade damaged D1.     

Costs and benefits of induced resistance in a clonal plant network

Plant defense theory suggests that inducible resistance has evolved to reduce the costs of constitutive defense expression. To assess the functional and potentially adaptive value of induced resistance it is necessary to quantify the costs and benefits associated with this plastic response. The ecological and evolutionary viability of induced defenses ultimately depends on the long-term balance between advantageous and disadvantageous consequences of defense induction. Stoloniferous plants can use their inter-ramet connections to share resources and signals and to systemically activate defense expression after local herbivory. This network-specific early-warning system may confer clonal plants with potentially high benefits. However, systemic defense induction can also be costly if local herbivory is not followed by a subsequent attack on connected ramets. We found significant costs and benefits of systemic induced resistance by comparing growth and performance of induced and control plants of the stoloniferous herb Trifolium repens in the presence and absence of herbivores.     

Reduced virus infectivity inN. tabacum secreting a TMV-specific full-size antibody

We developed a new concept of controlling plant virus infection based on the expression and secretion of full-size antibodies in plants. The neotope-specific anti-Tobacco Mosaic Virus (anti-TMV) antibody mAb24 has a high affinity towards epitopes present only on the surface of intact tobacco mosaic virions. The infectivity of the virus is inhibited almost completely if TMV is adsorbedin vitro at ratios as low as 300 antibody molecules per virion prior to inoculation. Cloned full-size cDNAs of mAb24 heavy and light chains were integrated into the plant expression vector pSS in tandem array and used for transformation ofNicotiana tabacum. The resulting transgenic tobacco plants expressed heavy- and light-chains of mAb24 which were assembled into functional antibodies and exported to the intercellular space. TMV specificity and affinity of the plant-produced antibody (mAb24-P) was not altered when compared to the original murine mAb24. F₁ progenies segregated 3:1 with respect to antibody secretion and showed up to two-fold higher expression levels compared to the F₀ plants. Upon infection with TMV F₁ plants producing mAb24-P showed a reduction of necrotic lesion numbers which is correlated with the amount of antibody produced in transgenic plants.The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X67210 (heavy-chain cDNA) and X67211 (light-chain cDNA).     

New World Bats Harbor Diverse Influenza A Viruses

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.     

Patterns of beta diversity in Europe: the role of climate,land cover and distance across scales

Aim We test the prediction that beta diversity (species turnover) and the decay of community similarity with distance depend on spatial resolution (grain). We also study whether patterns of beta diversity are related to variability in climate, land cover or geographic distance and how the independent effects of these variables depend on the spatial grain of the data. Location Europe, Great Britain, Finland and Catalonia. Methods We used data on European birds, plants, butterflies, amphibians and reptiles, and data on British plants, Catalonian birds and Finnish butterflies. We fitted two or three nested grids of varying resolutions to each of these datasets. For each grid we calculated differences in climate, differences in land‐cover composition (CORINE) and beta diversity (β_sim, β_Jaccard) between all pairs of grid cells. In a separate analysis we looked specifically at pairs of adjacent grid cells (the first distance class). We then used variation partitioning to identify the magnitude of independent statistical associations (i.e. independent effects in the statistical sense) of climate, land cover and geographic distance with spatial patterns of beta diversity. Results Beta diversity between grid cells at any given distance decreased with increasing grain. Geographic distance was always the most important predictor of beta diversity for all pairwise comparisons at the extent of Europe. Climate and land cover had weaker but distinct and grain‐dependent effects. Climate was more important at relatively coarse grains, whereas land‐cover effects were stronger at finer grains. In the country‐wide analyses, climate and land cover were more important than geographic distance. Climatic and land‐cover models performed poorly and showed no systematic grain dependence for beta diversity between adjacent grid cells. Main conclusions We found that relationships between geographic distance and beta diversity, as well as the environmental correlates of beta diversity, are systematically grain dependent. The strong independent effect of distance indicates that, contrary to the current belief, a substantial fraction of species are missing from areas with a suitable environment. Moreover, the effects of geographic distance (at continental extents) and land cover (at fine grains) indicate that any species distribution modelling should take both environment and dispersal limitation into account.     

CD4+CD25-Foxp3+ T cells: a marker for lupus nephritis?

Introduction

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease, which can affect different organs. Increased proportions of CD4⁺CD25^-Foxp3⁺ T cells have been described in SLE patients. The exact role of this cell population in SLE patients still remains unclear. We therefore analyzed this T cell subset in a large cohort of SLE patients with different organ manifestations.

Methods

Phenotypic analyses, proportions and absolute cell numbers of CD4⁺CD25^-Foxp3⁺ T cells were determined by flow cytometry (FACS) in healthy controls (HC) (n = 36) and SLE patients (n = 61) with different organ manifestations. CD4⁺CD25^-Foxp3⁺ T cells were correlated with clinical data, the immunosuppressive therapy and different disease activity indices. In patients with active glomerulonephritis, CD4⁺CD25^-Foxp3⁺ T cells were analyzed in urine sediment samples. Time course analyses of CD4⁺CD25^-Foxp3⁺ T cells were performed in patients with active disease activity before and after treatment with cyclophosphamide and prednisone.

Results

CD4⁺CD25^-Foxp3⁺ T cells were significantly increased in active SLE patients and the majority expressed Helios. Detailed analysis of this patient cohort revealed increased proportions of CD4⁺CD25^-Foxp3⁺ T cells in SLE patients with renal involvement. CD4⁺CD25^-Foxp3⁺ T cells were also detected in urine sediment samples of patients with active glomerulonephritis and correlated with the extent of proteinuria.

Conclusion

CD4⁺CD25^-Foxp3⁺ T cells resemble regulatory rather than activated T cells. Comparative analysis of CD4⁺CD25^-Foxp3⁺ T cells in SLE patients revealed a significant association of this newly described cell population with active nephritis. Therefore CD4⁺CD25^-Foxp3⁺ T cells might serve as an important tool to recognize and monitor SLE patients with renal involvement.     

Uptake of Amino Acids by Brain Microvessels Isolated from Rats after Portacaval Anastomosis

Abstract: The uptake of amino acids by microvessels isolated from brains of rats was studied. Previous studies have demonstrated alterations in blood-brain amino acid transport after portacaval shunt in rats. In order to elucidate whether such changes in the blood-brain barrier were located in the microvessels, brain microvessels were isolated from both rats with portacaval shunt and controls. Brain microvessels from rats 2 weeks after shunt operations took up significantly greater amounts of ¹⁴C-labeled neutral amino acids, but not of glutamic acid. lysine, or α-methylaminoisobutyric acid than microvessels from sham-operated controls. Measurement of uptake kinetics showed a higher V _max for phenylalanine and leucine uptake and a lower V _max for lysine uptake in microvessels from shunted rats compared with control, whereas the respective K _m's of uptake were similar in both preparations. The results suggest that changes in brain microvessel transport activity account for altered brain neutral amino acid concentrations after portacaval shunt and that such changes can be studied in vitro in isolated microvessels.