北极海洋链霉菌604F的卤化酶基因克隆及特征

【目的】本研究旨在克隆来自北极海洋、具有合成卤化物潜力的链霉菌(Streptomyces sp.)604F中的一个卤化酶基因,为后续克隆卤化物合成基因簇、分离鉴定卤化物提供指导。【方法】利用琼脂块法初步测试抗菌活性,借助液相色谱-飞行时间串联质谱技术(LC-Tof MS)初步寻找Streptomyces sp.604F发酵粗提液中的卤化物,并以简并PCR扩增合成次级代谢产物的指示基因(I型、II型聚酮合酶及非核糖体多肽合成酶编码基因);根据依赖黄素腺嘌呤二核苷酸的卤化酶基因保守区设计的简并引物,扩增基因片段并测序分析;采用高效热不对称交错PCR(hiTAIL-PCR)技术扩增卤化酶基因全长。【结果】Streptomyces sp.604F具有较强的抗白色念珠菌活性,其基因组同时含有编码I型聚酮合酶、II型聚酮合酶和非核糖体多肽合成酶基因,以及卤化酶基因;通过染色体步移克隆该卤化酶基因全长,共1443 bp,编码一个新的非色氨酸卤化酶,在合成已知卤化物的卤化酶数据库中,与其同源关系最近的为一类参与合成糖肽类次级代谢产物的卤化酶。【结论】Streptomyces sp.604F具有新的非色氨酸卤化酶,且推测参与糖肽类化合物的卤化修饰,为后续寻找目的卤化物提供了指导,也为研究该合成基因簇奠定基础。     

代谢组学方法鉴定球孢白僵菌孢子萌发和杀虫毒力相关的标记物

【目的】识别球孢白僵菌萌发和杀虫毒力相关的标记物。【方法】本研究对7个球孢白僵菌菌株的孢子进行萌发率和对茶毛虫毒力进行测定,采用基于LC-MS的代谢组学方法,识别菌丝和孢子提取物中与萌发和毒力有关的标记物。【结果】高毒力菌株的菌丝中具有高含量的carnitine、hercynine、acetylcarnitine、α,α-trehalose;Octa-Me、arg-arg-gln、phosphatidylethanolamine[PE(18∶2/0∶0)]、phosphotidylcholine[PC(18∶3/0∶0)]和PC(18∶2/0∶0)。高萌发率菌株的孢子中具有高含量的2,3-dimethylmaleate、acetylcarnitine、propionyl-carnitine和PC(18∶2/0∶0)。高毒力菌株的孢子中具有高含量的histamine、2,5-pyrrolidinedicarboxylic acid;Diamide、carnitine、acetylcarnitine、propionyl-carnitine、butyrylcarnitine、PE(18∶2/0∶0)、PC(16∶1/0∶0)和PC(18∶3/0∶0)。此外,对菌丝中杀虫相关的环肽beauverolides,beauvericins和bassianolide进行相对含量比较分析,发现单独一种肽的含量高低与菌株对茶毛虫的毒力没有直接关系,但高毒力的Bb1898菌株中的9种肽同时具有较高的含量暗示它们可能发挥协同作用。【结论】毒力和萌发的共同标记物是脂酰肉碱和磷脂,它们可能具有维持附着胞膨压和为穿透寄主提供能量的功能。其它标记物,如在高毒力菌丝中发现的hercynine和α,α-trehalose;高萌发率孢子中的2,3-dimethylmaleate,高毒力孢子中的histamine和2,5-pyrrolidinedicarboxylic acid Diamide,它们的作用原理有待进一步研究。     

NF-κB激活gp96转录从而促进肝细胞生长、细胞周期进展和细胞转化

【目的】探讨乙肝感染中gp96上调的机制及其可能发挥的病理学机制。【方法】首先通过生物信息学、Real-time PCR、荧光报告基因和Western blot研究NF-κB激活gp96表达的机制。进一步通过在肝细胞中过表达或敲低gp96的水平,运用CCK-8法和流式检测分析gp96对肝细胞增殖、凋亡,和细胞周期的影响,通过检测肝细胞EMT发生和细胞集落形成实验,分析gp96对于HCC发生的作用。【结果】NF-κB与gp96启动子上NF-κB结合位点结合,激活gp96的表达。实验结果显示,gp96能够促进肝细胞增殖、抑制凋亡,促进细胞周期从静息期向分裂期的转化,同时促进肝细胞EMT发生和细胞集落的形成。【结论】NF-κB通过活化gp96启动子上调其表达,为HBV慢性感染上调gp96的机制提供了线索,同时提示gp96在慢性炎症引发HCC过程中发挥重要作用。     

亚成体大熊猫肠道真菌多样性

【目的】分析5只亚成体大熊猫肠道真菌的多样性。【方法】采用基于ITS基因的RFLP(Restriction Fragment Length Polymorphism)方法,对肠道真菌总DNA进行ITS-RFLP分析,构建ITS克隆文库,然后用HhaI、HaeIII进行酶切指纹图谱分析,测序并绘制系统进化树。【结果】研究表明,5只亚成体大熊猫肠道真菌主要由Ascomycota(平均占46.24%)、Basidiomycota(平均占15.79%)2个门和一些未分类(平均占29.14%)、未培养(平均占8.83%)的真菌。其中,Ascomycota主要以Saccharomycetes(平均占63.74%)和Dothideomycetes(平均占35.91%)2个纲为主;Basidiomycota主要以Tremellomycetes(平均占65.80%)和Microbotryomycetes(平均占33.15%)2个纲为主。而这4个纲分别则主要以Candida、Debaryomyces;Pleosporales、Myriangium;Cystofilobasidium、Trichosporon;Leucosporidium、Leucosporidiella八个菌属为主,各样品中所占比例不等。【结论】亚成体大熊猫肠道内存在一定比例的真菌菌群,且ITS-RFLP技术能够很好地对其多样性进行分析。真菌的发现扩大了我们对大熊猫肠道微生物结构的了解,同时也有助于我们进一步研究真菌能否帮助大熊猫消化高纤维素食物。     

Microchip-based human serum atherogenic lipoprotein profile analysis

Owing to the mounting evidence of serum lipid changes in atherosclerosis, there has been increasing interest in developing new methods for analyzing atherogenic lipoprotein profiles. The separation of lipoprotein and lipoprotein subclasses has been demonstrated using a microchip capillary electrophoresis (CE) system [Chromatographia 74 (2011) 799–805]. In contrast to this previous study, the current report demonstrates that sdLDL peak efficiencies can be improved dramatically by adding gold nanoparticles (AuNPs) to the sample. Moreover, NBD C₆-ceramide was identified as a satisfactory dye for specific labeling and quantitation of individual serum lipoproteins. The accuracy of the method was evaluated by comparison with ultracentrifuge separated small, dense, low-density lipoprotein (sdLDL). A high correlation was observed between these two methods for sdLDL cholesterol. Lipid levels were investigated between atherosclerotic patients and healthy controls. The variation of serum atherogenic lipoprotein profiles for atherosclerotic patients pre- and post-treatment was assessed by microchip CE. This method has potential for the rapid and sensitive detection of different lipoprotein classes as well as their subclasses and, therefore, is suitable for routine clinical applications. Microchip-based atherogenic lipoprotein profile assays will greatly improve the analysis of risk factors in atherosclerosis and will provide useful information for monitoring the effect of therapies on atherosclerotic disease.     

Bacillus cihuensis sp. nov., isolated from rhizosphere soil of a plant in the Cihu area of Taiwan

A Gram-positive, moderately halotolerant, rod-shaped, spore forming bacterium, designated strain FJAT-14515^T was isolated from a soil sample in Cihu area, Taoyuan County, Taiwan. The strain grew at 10–35 °C (optimum at 30 °C), pH 5.7–9.0 (optimum at pH 7.0) and at salinities of 0–5 % (w/v) NaCl (optimum at 1 % w/v). The diagnostic diamino acid of the peptidoglycan of the isolated strain was meso-diaminopimelic acid and major respiratory isoprenoid quinone was MK-7. Major cellular fatty acids were anteiso-C_15:0 (40.6 %), iso-C_15:0 (20.7 %) and the DNA G+C content of strain FJAT-14515^T was 37.1 mol %. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-14515^T belongs to the genus Bacillus, and was most closely related to the reference strains of Bacillus muralis DSM 16288^T (97.6 %) and Bacillus simplex DSM 1321^T (97.5 %). Levels of DNA–DNA relatedness between strain FJAT-14515^T and the reference strains of B. muralis DSM 16288^T and B. simplex DSM 1321^T were 27.9 % ± 3.32 and 44.1 % ± 0.57, respectively. Therefore, on the basis of phenotypic, chemotaxonomic and genotypic properties, strain FJAT-14515^T represents a novel species of the genus Bacillus, for which the name Bacillus cihuensis sp. nov. is proposed. The type strain is FJAT-14515^T (=DSM 25969^T = CGMCC 1.12697^T).     

Bacillus bingmayongensis sp. nov., isolated from the pit soil of Emperor Qin’s Terra-cotta warriors in China

A Bacillus-like isolate, strain FJAT-13831^T, isolated from the No. 1 pit soil of Emperor Qin’s Terra-cotta Warriors in Xi’an City, China, was studied to determine its taxonomic status. Dominant fatty acids of this organism included iso-C_15:0, iso-C_17:0, C_16:0, iso-C_13:0, anteiso-C_15:0, and iso-C_17:1ω5c. Comparative 16S rRNA gene sequence analysis confirmed the affiliation of this isolate to the genus Bacillus and indicated that it was closely related to Bacillus pseudomycoides DSM 12442^T (99.72 % similarity). A phylogenetic analysis of the gyrB gene sequence similarities exhibited independent clustering of the isolate FJAT-13831^T and showed 93.8 % (<95 %) sequence similarity with its closest phylogenetic neighbour B. pseudomycoides DSM 12442^T. Separate standing of the strain FJAT-13831^T was supported by a whole genome-based phylogenetic analysis with an average nucleotide identity value of 91.47 (<95 %) between isolate FJAT-13831^T and B. pseudomycoides DSM 12442^T and was consistent with the results of DNA–DNA hybridization (69.1 % relatedness). These findings support the conclusion that the isolate FJAT-13831^T represents a novel species, for which the name Bacillus bingmayongensis sp. nov. is proposed. The type strain is FJAT-13831^T (= CGMCC 1.12043^T = DSM 25427^T).     

Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children

The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric population, to further clarify its significance in causing inflammation of gastric mucosa in children and to verify whether cagA, vacA, iceA and babA genes work independently or synergistically in causing gastritis. A total of 163 H. pylori isolates obtained from the same number of children were tested for the presence of cagA, vacA and iceA genes using previously established methods, while the babA2 gene was determined using novel polymerase chain reaction assay targeting a 139-bp fragment of the central region of babA2. The babA2 gene was detected in 47.9 % of H. pylori samples. The presence of the babA2 gene was strongly associated with cagA, vacA s1 and vacA m1 genotype. The babA2 status correlated positively with bacterial density score, activity of inflammation and chronic inflammation of gastric mucosa. No significant correlation was found between the babA2 status and the presence of atrophy or intestinal metaplasia. In addition, the activity of gastric inflammation and density score were significantly associated with the coexpression of the cagA, vacA s1, vacA m1 and babA2 genes. The study, which included the largest number of pediatric H. pylori samples to date, confirmed that babA2 gene plays an important role in the pathogenesis of H. pylori gastritis in children. Furthermore, our results suggest that babA2, cagA and vacA s1 and m1 gene products may work synergistically in worsening the inflammation of gastric mucosa.     

Dynamics of apelin receptor/G protein coupling in living cells

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi₂ or Gαi₃ through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi₁ subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi₂, Gαi₃, Gαo, Gαq; among which Gαi₂, Gαi₃ were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.