IL-15 participates in the respiratory innate immune response to influenza virus infection

Following influenza infection, natural killer (NK) cells function as interim effectors by suppressing viral replication until CD8 T cells are activated, proliferate, and are mobilized within the respiratory tract. Thus, NK cells are an important first line of defense against influenza virus. Here, in a murine model of influenza, we show that virally-induced IL-15 facilitates the trafficking of NK cells into the lung airways. Blocking IL-15 delays NK cell entry to the site of infection and results in a disregulated control of early viral replication. By the same principle, viral control by NK cells can be therapeutically enhanced via intranasal administration of exogenous IL-15 in the early days post influenza infection. In addition to controlling early viral replication, this IL-15-induced mobilization of NK cells to the lung airways has important downstream consequences on adaptive responses. Primarily, depletion of responding NK1.1+ NK cells is associated with reduced immigration of influenza-specific CD8 T cells to the site of infection. Together this work suggests that local deposits of IL-15 in the lung airways regulate the coordinated innate and adaptive immune responses to influenza infection and may represent an important point of immune intervention.     

5-Sulfonyl-benzimidazoles as selective CB2 agonists

A novel series of benzimidazole CB2-receptor agonists was synthesized and the structure-activity relationship explored. The results showed agonistic activities with an EC(50) up to 0.5 nM and excellent selectivity (>4000-fold) over the CB1 receptor. The size of the substituent on the 2-position determined the level of agonism, ranging from inverse agonism to partial agonism to full agonism, which was more pronounced for the rat CB2 receptor. A wide variation of sulfonyl substituents at the benzimidazole 5-position was tolerated, which was used to optimize the drug-like properties. This resulted into lead compound 14j that can be used to investigate the potential of a selective, peripherically acting CB2 agonist. The in vitro profile of key compounds is displayed using pie bar charts (VlaaiVis).     

AtCDKA;1 silencing in Arabidopsis thaliana reduces reproduction of sedentary plant-parasitic nematodes

SUMMARY: The activity of the Arabidopsis thaliana cyclin-dependent kinase AtCDKA;1 is important throughout G(1)/S and G(2)/M transitions and guarantees the progression of the cell cycle. Inhibitor studies have shown that activation of the cell cycle is important for the development of nematode feeding sites. The aim of this study was to silence the expression of the AtCDKA;1 gene in nematode feeding sites to interfere with their development. Therefore, sense and antisense constructs were made for the AtCDKA;1 gene and fused to a nematode-inducible promoter which was activated in nematode feeding sites at an earlier time point than AtCDKA;1. Two transgenic A. thaliana lines (S266 and S306) containing inverted repeats of the AtCDKA;1 gene and with reduced AtCDKA;1 expression in seedlings and galls were analysed in more detail. When the lines were infected with the root-knot nematode Meloidogyne incognita, significantly fewer galls and egg masses developed on the roots of the transgenic than wild-type plants. Infection of the AtCDKA;1-silenced lines with Heterodera schachtii resulted in significantly fewer cysts compared with controls. The S266 and S306 lines showed no phenotypic aberrations in root morphology, and analysis at different time points after infection demonstrated that the number of penetrating nematodes was the same, but fewer nematodes developed to maturity in the silenced lines. In conclusion, our results demonstrate that silencing of CDKA;1 can be used as a strategy to produce transgenic plants less susceptible to plant-parasitic nematodes.     

人肿瘤坏死因子β(hTNFβ)在变铅青链霉菌中的表达研究

以变铅青链霉菌为宿主研究了人INFβ(hTNFβ)的异源表达。应用链霉菌S.VENEZUELAC cbs762.70分泌产生的枯草杆菌蛋白酶抑制剂vsi基因的启动子、表达调控序列和分泌信号肽序列,分别对hTNFβ进行了直接分泌表达、分泌融合表达和胞内表达。将hTNFβ的cDNA分别直接融合于vsi信号肽序列下游2个氨基酸处、vsi全长基因之后以及vsi起始密码子ATG的下游,获得的表达盒分别克隆至链霉菌高拷贝质粒pIJ486,转化Streptomyces lividans TK24,获得了重组菌株S.lividans(pIJ486-hTNFβ),s.LIVIDANS(PIJ486-vsi-hTNFβ)和S.lividans(pIVPA-hTNFβ)。分别对不同的重组菌株进行摇瓶培养,对其培养的上清液和细胞裂解液进行SDS—PAGE和Westen杂交,结果表明：hTNFβ在重组菌株中均获得了表达,且直接分泌产物和胞内表达产物均具有生物学活性。hTNFβ直接分泌表达产物的分子量约为16kDa,NB培养基中培养48h时表达水平约为0．7mg／L。胞内表达产物分子量与对照重组hTNFβ一致(18．7kDa),但随培养时间的延长远步降解为16kDa,NB培养基中培养48h时的表达水平(25．1mg／L)远高于其直接分泌表达水平。     

An insight into critical endocycle genes for plant-parasitic nematode feeding sites establishment

Root-knot and cyst nematodes are biotrophic parasites that invade the root apex of host plants and migrate toward the vascular cylinder where they cause the differentiation of root cells into galls (or root-knots) containing hypertrophied multinucleated giant-feeding cells, or syncytia, respectively. The precise molecular mechanisms that drive the formation of such unique nematode feeding sites are still far-off from being completely understood. The diverse gene expression changes occurring within the host cells suggest that both types of plant-parasitic nematodes modulate a variety of plant processes. Induction and repression of genes belonging to the host cell cycle control machinery have shown to be essential to drive the formation of such specialized nematode feeding cells. We demonstrate that nematodes usurp key components regulating the endocycle in their favor. This is illustrated by the involvement of anaphase-promoting complex (APC) genes (CCS52A and CCS52B), the endocycle repressor DP-E2F-like (E2F/DEL1) gene and the ROOT HAIRLESS 1 PROTEIN (RHL1), which is part of a multiprotein complex of the toposiomerase VI, in the proper formation of nematode feeding sites. Altering the expression of these genes in Arabidopsis plants by down- or overexpressing strategies strongly influences the extent of endoreduplication in both types of nematode feeding site leading to a disturbance of the nematode’s life cycle and reproduction.     

Effect of T-DNA configuration on transgene expression

Summary T-DNA vectors were constructed which carry a -glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3 end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for -glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Molecular phylogeny and evolution of Caricaceae based on rDNA internal transcribed spacers and chloroplast sequence data

This study focused on clarifying phylogenetic relationships and evolution within Caricaceae. Our phylogenetic analysis based on nucleotide sequences from the ITS of the ribosomal DNA and three chloroplast fragments (matK, trnL-trnF, and psbA-trnH) included 29 taxa belonging to five genera: the neotropical genera Carica, Vasconcellea, Jarilla, and Jacaratia and the equatorial African genus Cylicomorpha. Having a relatively low mutation rate, matK, and trnL-trnF were used for estimating relationships at the generic level, while intrageneric evolution within Vasconcellea was studied with the more variable ITS and psbA-trnH sequences. Gaps, coded as binary characters, were added to the sequence alignments before performing Maximum Parsimony and Maximum Likelihood analyses. Monophyly of Caricaceae as well as phylogenetic distance between Carica and Vasconcellea species, previously belonging to the same genus, and monophyly of the resurrected genus Vasconcellea were emphasized. Within Vasconcellea, the largest genus of this family, two well-confirmed evolutionary lineages could be discerned: (1) V.xheilbornii, V. weberbaueri, V. stipulata, and V. parviflora and (2) a clade holding all other taxa of the genus. Incongruence between nuclear ITS and chloroplast psbA-trnH datasets, shown to be significantly caused by some taxa of the genus Vasconcellea, indicated that reticulate events in this genus might be more frequent than previously suspected. Moreover, intra-individual ITS sequence heterogeneity provided further evidence for the hybrid or introgressed origin of different taxa and one presumed hybrid belonging to this genus.