脂环酸芽孢杆菌D-1的耐盐内切葡聚糖酶基因克隆、表达与酶学性质北大核心CSCD

【目的】克隆温泉中嗜热嗜酸的脂环酸芽孢杆菌D-1(Alicyclobacillus tengchongensis CGMCC1504)的内切葡聚糖酶基因gluE1,并对该酶进行序列分析和重组酶的酶学特性分析。【方法】通过全基因组测序获得gluE1全长,并对其氨基酸序列(GluE1)进行分析。将gluE1重组到载体p EASY-E2中并转化到大肠杆菌BL21(DE3)中异源表达,利用组氨酸标签纯化GluE1并进行酶学性质分析。【结果】gluE1与NCBI数据库中GH5的内切葡聚糖酶具有较高的相似性,全长1020 bp,GC含量50.5%,编码339个氨基酸(40.45 k Da)。GluE1与数据库中序列的最高一致性为97%,与其余纤维素酶的一致性<60%。GluE1可水解CMC-Na、可溶性淀粉和大麦β-葡聚糖,表观最适pH为6.5,pH 5.0–10.0稳定并维持60%以上的酶活性。GluE1的表观最适温度为55℃,在37℃下稳定。在55℃ pH 6.5条件下,GluE1对大麦β-葡聚糖的K_m、V_(max)和k_(cat)分别为8.58 mg/mL、416.67 U/mg和280.90 s^(–1)。GluE1受Ag^+、Hg^(2+)及SDS抑制,β-巯基乙醇、Pb^(2+)、Mg^(2+)、Ca^(2+)和Na^+对GluE1有微弱的促进作用,NaCl对GluE1的影响不大,加入30%的NaCl,仍有64%以上的酶活性;经30%的NaCl在37℃下处理60 min,仍能保持93%以上的活性。【结论】首次报道从Alicyclobacillus属的细菌中克隆得到内切葡聚糖酶基因并对其酶学性质进行研究,GluE1具有良好的pH稳定性和有较强的耐盐性,可能具有更大应用潜力。     

油樟悬浮培养及次生代谢产物的诱导

以油樟(Cinnamomum longepaniculatum)叶片为外植体在附加6-BA和NAA不同激素浓度组合的MS培养基上筛选质地疏松、生长旺盛的愈伤组织, 分别接种在MS、B5、WPM三种液体培养基中进行细胞悬浮培养, 并检测诱导产生的次生代谢产物。结果表明: 2 mg.L-1 6-BA+ 0.5 mg.L-1 NAA能诱导质地疏松、生长旺盛的愈伤组织, B5基本培养基中细胞长势最好; 愈伤组织在B5+2 mg.L-1 6-BA+ 0.5 mg.L-1 NAA中悬浮培养, 继代2次后形成均一的单细胞; 从油樟悬浮培养物中检测出50%以上的成分是苯甲醇。     

中国西藏种子植物区系新资料

本文报道了采自西藏喜马拉雅南坡的8个中国种子植物新记录种以及1个西藏新记录属。前者分别是吉隆牛奶菜(Marsdenia roylei)、塔基棕榈(Trachycarpus takil)、喀西蜂斗草(Sonerila khasiana)、旋花锡生藤(Cissampelos convolvulacea)、吉隆角盘兰(Herminium edgeworthii)、尼泊尔西番莲(Passiflora napalensis)、椭穗姜花(Hedychium ellipticum)和藏南象牙参(Roscoea brandisii); 1个西藏新记录属为箭药藤属(Belostemma) (箭药藤 Belostemma hirsutum)。凭证标本存放于中国科学院西双版纳热带植物园标本馆(HITBC)和西藏自治区高原生物研究所标本室(XZ)。     

一株耐盐葡萄球菌的分离鉴定及其特性分析

本文旨在分离筛选出在高盐环境中具有潜在应用可能性的菌株。在筛选耐盐酵母的过程中分离出一株耐盐菌株,采用形态特征观察、生理生化实验和16S rDNA基因测序进行菌株鉴定,鉴定结果显示该菌为沃氏葡萄球菌(Staphylooccus warneri),命名为SW-1。进一步对其药敏特性、耐盐性和表面疏水性进行检测,结果表明,该菌株对大部分抗生素极敏感,在含20%(质量分数)NaCl的LB培养基中仍能良好生长,对氯仿、乙酸乙酯和正丁醇的疏水率依次为43%、34%和39%。沃氏葡萄球菌SW-1在高盐条件下能快速适应环境积累生物量,具有优良的生物学特性,为开发和利用耐盐菌株提供参考。     

Structures and biological activities of three synaptic antagonists from orb weaver spider venom

The venom of Argiope aurantia, an orb weaver spider, contains a mixture of low molecular weight "argiotoxins", which block neuromuscular transmission in insects. Complete structure elucidation of three argiotoxins reveals common features; a hydrophilic, basic domain of arginine, a polyamine and asparagine is connected to an aromatic moiety contributed either by 4-hydroxyindole-3-acetic acid or 2,4-dihydroxyphenylacetic acid. Structural assignments of two argiotoxins are verified by chemical synthesis. The argiotoxins cause reversible paralysis when injected into insects and this is correlated with a stimulus-dependent inhibition of skeletal neuromuscular transmission at submicromolar concentrations.     

Suppression of glutathione S-transferases in rat seminal vesicles or pituitary glands

We have studied the tissue-specific expression of GSH S-transferases in rat seminal vesicles and pituitary glands by in vitro translation and immunoprecipitation. The major GSH S-transferase subunit expressed in rat seminal vesicles belongs to the Yb mobility class whose expression diminishes when the rats are treated with pentobarbital. The pattern of GSH S-transferase expression in the pituitary gland is very similar to that of the rat brain with Yb size subunit(s) predominant. The Y beta size subunit is also expressed together with the Yc and Y delta subunits. The expression of GSH S-transferases was drastically reduced in pituitary gland poly(A) RNAs from diethylstilbestrol-treated, ovariectomized female rats. Xenobiotics such as phenobarbital, 3-methylcholanthrene, and trans-stilbene oxide induce rat liver GSH S-transferase activities, especially the Ya- and Yb-subunit containing isozymes. Induction of GSH S-transferases by a combination of the three xenobiotics is neither additive nor synergistic, however. Our results clearly demonstrate that GSH S-transferase expression in seminal vesicles and pituitary glands can be suppressed by phenobarbital and diethylstilbestrol, respectively. Our findings suggest that different GSH S-transferase isozymes respond differently to various xenobiotics. Both induction and suppression occur in rats treated with xenobiotics. This notion helps to explain the lack of additive or synergistic induction in rats treated with more than one xenobiotic.     

A unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide

A novel type of enzyme which hydrolyzes the linkage between the ceramide and the sugar chain in various glycosphingolipids has been found in the leech, Hirudo medicinalis. This enzyme releases the intact oligosaccharide from LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, nLcOse4Cer, GM3, GM2, GM1, GD1a and GT1 with the concurrent release of ceramides. By using tritium-labeled GM1 as substrate we found the optimum pH of this enzyme to be between pH 4 and 5. Since the enzyme cleaves the linkage between the ceramide and the sugar chain in various glycosphingolipids with no apparent preference toward the sugar chain, we propose to call this enzyme ceramide-glycanase.     

In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of serious hemorrhagic diseases in humans with high mortality rates. CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen. However, the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing. We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain, IbAr10200, which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8 ?. The structure confirms that CCHFV Gc is a class II fusion protein. Unexpectedly, structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain III from phenuiviruses, suggesting a complex evolutionary pathway among these bunyaviruses. Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure, providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.     

Development of a fluorescent probe hydrolysis-insulated isothermal PCR for rapid and sensitive on-site detection of African swine fever virus

Highlights
1. A probe-based insulated isothermal PCR (iiPCR) assay was developed for rapid and onsite detection of ASFV.
2. The developed iiPCR showed similar sensitivity and specificity with OIE recommended real-time PCR.
3. Blood samples could be directly applied as PCR template in iiPCR without DNA extraction.