A novel Meloidogyne graminicola effector,MgMO237, interacts with multiple host defence‐related proteins to manipulate plant basal immunity and promote parasitism

Plant‐parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up‐regulated in parasitic third‐/fourth‐stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two‐hybrid and co‐immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3‐β‐glucan synthase component (OsGSC), cysteine‐rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis‐related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence‐related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence‐related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes.     

Potentiation of antibiotic activity by Passiflora cincinnata Mast. front of strains Staphylococcus aureus and Escherichia coli

The development of new drugs from plants is an interesting alternative approach to overcoming microbial resistance. Passiflora cincinnata shows resistance to diseases and pests and a higher concentration of chemical components that may be useful in the pharmaceutical industry. We investigated the potential antimicrobial and antibiotic-modifying activity of hydroalcoholic extracts of leaves, stems, bark, pulp and seeds of P. cincinnata. The extracts were prepared by homogenization of material in 50% ethanol. Minimum inhibitory concentration (MIC) was determined by the broth dilution method, and the bacterial strains tested were Staphylococcus aureus and Escherichia coli. Antibiotic-modifying activity was evaluated against the strains S. aureus 03 and E. coli 08, using a subinhibitory concentration of extract. The antibiotics tested were: amikacin, gentamicin, ampicillin, potassium benzylpenicillin and oxacillin. The extracts did not show antimicrobial activity of clinical relevance, where the MIC was equal to or greater than 1024 μg/mL. S. aureus showed 13 events, while E. coli showed only 4 events. Among these events, 14 involved synergistic activity, potentiating the effect of the antibiotics, and only 3 events demonstrated antagonistic activity toward ampicillin. Hydroalcoholic extracts are potential antimicrobial agents when combined with conventional drugs little utilized in in vivo treatment.     

Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl^? channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.     

人源CT55蛋白原核表达及单克隆抗体的制备

为制备Cancer testis 55(CT55)单克隆抗体,需构建带有人源CT55片段的原核表达质粒,把该质粒转化Rosetta感受态进行原核表达并得到目的蛋白,蛋白质被纯化后免疫6周雌性BALB/c小鼠。按传统的单克隆抗体的制备方法,取小鼠脾细胞与骨髓瘤细胞(sp2/0)进行融合,经ELISA方法筛选及两次连续亚克隆,共获得多株能稳定分泌抗CT55蛋白单克隆抗体的杂交瘤细胞,如3D8B7B12、4C8E1C9、3D8C10G9等。ELISA及Western blot(WB)分析结果表明,筛选的细胞株均能产生单克隆抗体,且该抗体均分别能与原核表达及真核表达的CT55蛋白发生特异性结合。单克隆抗体可用于免疫荧光试验,且与P53发生互作的荧光主要位于细胞核边缘。结果表明,成功制备了针对人源CT55蛋白的单克隆抗体。CT55蛋白单克隆抗体的制备为今后肝癌、胃癌、结肠癌等癌症的快速的病原学诊断以及CT55蛋白的结构和功能研究奠定了物质基础。     

Characterization of the macronuclear DNA of different species ofTetrahymena

Summary The macronuclear DNAs from 20 different species ofTetrahymena were characterized using alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100–2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species ofTetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species ofTetrahymena based on this hybridization pattern paralels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains ofTetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells

Summary A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold from cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was CAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles ³²P, per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its K_m was 30 M. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnC12 and 4- to 9-fold by deoxymononucleotides.The nuclei of HeLa cells contained 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a CAMP-independent protein kinase which is similar to glycogen synthase kinase.Abbreviations RNP ribonucleoprotein - PMSF phenyl methyl sulfonyl fluoride - EGTA ethylene glycol-bis-(-aminoethyl ether) N,N-tetra acetic acid - TCA trichloroacetic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay     

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt‐Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT‐TTR and L55P‐TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P‐TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single α‐helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation‐prone conformations characterized by full displacement of strands C and D from the main β‐sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H‐bond network and consequent destabilization of the CBEF β‐sheet of the β‐sandwich; (v) WT forms aggregation‐compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P‐TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.     

The effect of antiestrogens on egg yolk protein synthesis and estrogen-binding to chromatin in the rooster liver

Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [³H] estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.     

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB₁ receptor antagonist Rimonabant®, CB₂ receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB₁ receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB₂ ligand {"type":"entrez-nucleotide","attrs":{"text":"GW405833","term_id":"288331434","term_text":"GW405833"}}GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB₂ receptor ligands. The phytocannabinoids Δ⁹-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.