Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release.

The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand. These structures, together with biochemical data, reveal that proteolytic cleavage opens the extracellular domain of hDisp1, removing steric hindrance to Shh binding. Structure-guided functional experiments demonstrate the role of hDisp1-Shh interactions in ligand release. Our results clarify the mechanisms of hDisp1 activation and Shh morphogen release, and highlight how a unique proteolytic cleavage event enabled acquisition of a protein substrate by a member of a family of small molecule transporters.

Value of base excess in predicting the prognosis of patients with paraquat poisoning / 中华劳动卫生职业病杂志

Objective@#To explore prognostic factors of acute paraquat poisoning (APP) , analyze the correlation between base excess (BE) and plasma concentration of paraquat (C-PQ) and discuss BE level in evalua-tion of prognosis of acute paraquat poisoning patients.@*Methods@#We retrospectively selected 84 APP patients who admitted to Emergency Intensive Care Unit (EICU) of our hospital from 2009.9.1 to 2015.8.31.Clinical data from 84 APP patients were analyzed. BE、C-PQ、time of gastric lavagesince ingestion、time of hemoperfusion since ingestion、severity index of paraquat poisoning (SIPP) 、white blood cell (WBC) 、percentage of neutrophils (N%) 、hemoglobin (HB) 、creatinine (Cr) 、alanine aminotransferase (ALT) 、aspartate aminotransferase (AST) 、partial pressure of oxygen (PaO2) 、partial pressure of carbon dioxide (PaCO2) and other laboratory parameters were measured. A total of 41 patients in non-survivors died during the 30 days after admission and 43 patients in survivors survived during the 30 days. The factors of prognosis in paraquat poisoining and the role of BE in evalu-ating prognosis was analyzed, as well as the correlation between BE and C-PQ.@*Results@#1.Logistic regression analyses showed BE、C-PQ、ALT、AST、Cr was of prognostic significance[odds ratio (OR) of BE: 0.511, 95%CI 0.267, 0.978; C-PQ:-=0.999, 95%CI 0.999, 1.000; both P<0.05] ; 2.The area under the receiver operating characteristic curve (ROC curve) of BE、C-PQ and prognosis were 0.775、0.927 respectively, BE≤-1.7 mmol/L was the best cut-off value, the sensitivity、specificity for predicting were 82.9%、62.8%, the evaluation value was lower to C-PQ>3 273.935 ng/ml (AUC 0.927, 78.0%、95.3%) ; 3.BE negative correlated with C-PQ[-1.100 (-4.100, -0.200) , -5.900(-8.650, -2.500) , both P<0.05]. (r=-0.4, P<0.01).@*Conclusion@#These results suggest that BE may be useful for the prediction of prognosis in PQ poisoning and BE negative correlated with C-PQ.

Comparative analyses of secreted proteins from the phytopathogenic fungus Verticillium dahliae in response to nitrogen starvation.

The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron-proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.

Streptomyces coelicolor SCO4226 is a nickel binding protein.

The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five ß-strands flanked by two α-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification.

Structural and biochemical analyses of Microcystis aeruginosa O-acetylserine sulfhydrylases reveal a negative feedback regulation of cysteine biosynthesis.

O-acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis from O-acetylserine (OAS) and inorganic sulfide in plants and bacteria. Bioinformatics analyses combined with activity assays enabled us to annotate the two putative genes of Microcystis aeruginosa PCC 7806 to CysK1 and CysK2, which encode the two 75% sequence-identical OASS paralogs. Moreover, we solved the crystal structures of CysK1 at 2.30Ǻ and cystine-complexed CysK2 at 1.91Ǻ, revealing a quite similar overall structure that belongs to the family of fold-type II PLP-dependent enzymes. Structural comparison indicated a significant induced fit upon binding to the cystine, which occupies the binding site for the substrate OAS and blocks the product release tunnel. Subsequent enzymatic assays further confirmed that cystine is a competitive inhibitor of the substrate OAS. Moreover, multiple-sequence alignment revealed that the cystine-binding residues are highly conserved in all OASS proteins, suggesting that this auto-inhibition of cystine might be a universal mechanism of cysteine biosynthesis pathway.

Identification and analysis of the germin-like gene family in soybean.

BACKGROUND: Germin and germin-like proteins constitute a ubiquitous family of plant proteins. A role of some family members in defense against pathogen attack had been proposed based on gene regulation studies and transgenic approaches. Soybean (G. max L. Merr.) germin genes had not been characterized at the molecular and functional levels. RESULTS: In the present study, twenty-one germin gene members in soybean cultivar 'Maple Arrow' (partial resistance to Sclerotinia stem rot of soybean) were identified by in silico identification and RACE method (GmGER 1 to GmGER 21). A genome-wide analyses of these germin-like protein genes using a bioinformatics approach showed that the genes located on chromosomes 8, 1, 15, 20, 16, 19, 7, 3 and 10, on which more disease-resistant genes were located on. Sequence comparison revealed that the genes encoded three germin-like domains. The phylogenetic relationships and functional diversity of the germin gene family of soybean were analyzed among diverse genera. The expression of the GmGER genes treated with exogenous IAA suggested that GmGER genes might be regulated by auxin. Transgenic tobacco that expressed the GmGER 15 [corrected] gene exhibited high tolerance to the salt stress. In addition, the GmGER mRNA increased transiently at darkness and peaked at a time that corresponded approximately to the critical night length. The mRNA did not accumulate significantly under the constant light condition, and did not change greatly under the SD and LD treatments. CONCLUSIONS: This study provides a complex overview of the GmGER genes in soybean. Phylogenetic analysis suggested that the germin and germin-like genes of the plant species that had been founded might be evolved by independent gene duplication events. The experiment indicated that germin genes exhibited diverse expression patterns during soybean development. The different time courses of the mRNAs accumulation of GmGER genes in soybean leaves appeared to have a regular photoperiodic reaction in darkness. Also the GmGER genes were proved to response to abiotic stress (such as auxin and salt), suggesting that these paralogous genes were likely involved in complex biological processes in soybean.

Researches and applications on pesticides from Chinese medicine plant origin / 中国中药杂志

The research progress on Chinese medicine plant resources with pesticide activities, the active components and their reaction mechanism as well as the application and prospect were reviewed in this paper. Some proposals on the exploitation of traditional Chinese medicine plant origin pesticide were given. It is suggested to found compounds with pesticide activities from heat clearing and toxic clearing medicinal plants.

Studies on the cell growth, differentiation and terpene lactone accumulation in Ginkgo biloba cell suspension cultures / 生物工程学报

To provide supports for Ginkgo biloba cell engineering for production of Terpene lactones (Ginkgolides and bilobalide), the cell suspension were established from calli induced from zygote embryos and stems of 30-day-old seedlings respectively. The relationship between cell growth, differentiation and the terpene lactone accumulation in these suspension cultures were investigated. HPLC determination indicated that, the ginkgolide B was found in the embryo derived cell suspension cultures at 0.044% of cell dry weight, and this result was the first time reported in this study. The accumulation of terpene lactone in the suspension cultures derived from both the embryo and seedling stems are effected by the level of the cell differentiation. The ginkgolide B was only found in small cell aggregates in the size smaller than 2mm, and the highest level of ginkgolide B was accumulated in cell aggregates in the size smaller than 1mm; however, the cell aggregates in the size bigger than 3mm could only produced bilobalide and ginkgolide A. In the same size aggregates of the suspension cultures the terpene lactone accumulation is strongly effected by the source of the explant. When the size of cell aggregates was in less than 1mm, the concentration of bilobalide, ginkgolide A and B in the cell suspension cultures derived from the embryos was 2, 1.4 and 0.56-fold, respectively, higher than that of cell cultures derived from seedling stems.