AvrRps4 effector family processing and recognition in lettuce

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector‐triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N‐terminus (AvrRps4^N) and the C‐terminus (AvrRps4^C). AvrRps4^C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4‐mediated immunity in Arabidopsis; on the other hand, AvrRps4^N induces HR in lettuce. Furthermore, AvrRps4^N‐mediated HR requires a conserved arginine at position 112 (R112), which is also important for full‐length AvrRps4 (AvrRps4^F) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO‐mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.     

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 microg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC50 values: HL-60 (147.3 +/- 15.2 microg/ml), Raji (253.8 +/- 60.7 microg/ml) and NB-4 (269.3 +/- 12.4 microg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC50 > 800 microg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.     

Three-dimensional gas exchange pathways in pome fruit characterized by synchrotron x-ray computed tomography

Our understanding of the gas exchange mechanisms in plant organs critically depends on insights in the three-dimensional (3-D) structural arrangement of cells and voids. Using synchrotron radiation x-ray tomography, we obtained for the first time high-contrast 3-D absorption images of in vivo fruit tissues of high moisture content at 1.4-microm resolution and 3-D phase contrast images of cell assemblies at a resolution as low as 0.7 microm, enabling visualization of individual cell morphology, cell walls, and entire void networks that were previously unknown. Intercellular spaces were always clear of water. The apple (Malus domestica) cortex contains considerably larger parenchyma cells and voids than pear (Pyrus communis) parenchyma. Voids in apple often are larger than the surrounding cells and some cells are not connected to void spaces. The main voids in apple stretch hundreds of micrometers but are disconnected. Voids in pear cortex tissue are always smaller than parenchyma cells, but each cell is surrounded by a tight and continuous network of voids, except near brachyssclereid groups. Vascular and dermal tissues were also measured. The visualized network architecture was consistent over different picking dates and shelf life. The differences in void fraction (5.1% for pear cortex and 23.0% for apple cortex) and in gas network architecture helps explain the ability of tissues to facilitate or impede gas exchange. Structural changes and anisotropy of tissues may eventually lead to physiological disorders. A combined tomography and internal gas analysis during growth are needed to make progress on the understanding of void formation in fruit.     

<Emphasis Type="Italic">Narcissus tazetta</Emphasis> lectin shows strong inhibitory effects against respiratory syncytial virus,influenza A (H1N1, H3N2, H5N1) and B viruses

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC₅₀ of 2.30 μg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC₅₀ values ranging from 0.20 μg/ml to 1.33 μg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC₅₀/IC₅₀≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.     

Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis

Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal‐cysteine‐protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL‐mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non‐eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL‐mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non‐eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL‐stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL‐stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL‐primed osteoclastogenesis was observed in male and female CatB‐knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL‐primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.     

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.