北京西山侏罗纪大同锥叶蕨的研究

大同锥叶蕨Coniopteris tatungensis 是侏罗纪植物群常见的分子。由于对该植物特征认识的不同, 对其在分类上的归属有不同意见,或归入膜蕨型锥叶蕨C．hymenophylloides,或归入简单锥叶蕨C. simplex。通过对产自北京西山斋堂地区窑坡组下段的标本的深入研究,特别是用扫描电子显微镜对该 标本的孢子囊群和孢子囊的观察,确认大同锥叶蕨在锥叶蕨属的种级分类上的独立性。文中还初步讨论了其分类和地理分布。     

水稻白叶枯病广谱抗性基因Xa21导入两用不育系培矮64S

以克隆的Xa21基因为外源基因,成熟胚愈伤组织为转化受体,应用农杆菌介导法对水稻两用型核不育系培矮64S进行转化,获46株转基因植株。PCR和Southern分析结果表明,Xa21已整合到受体基因组。用稻白叶枯病病原菌(Xanthomonasoryzaepv.oryzae)菲律宾小种6号接种鉴定,结果表明大多数转基因植株获得了抗病性。已整合的Xa21基因能够稳定地遗传,在所检测转基因株系的T1代中,Xa21基因显示3:1的分离。     

脓肿分枝杆菌经Toll样受体2介导的JNK/ERK信号通路诱导巨噬细胞表达肿瘤坏死因子α和白细胞介素8

为探讨脓肿分枝杆菌脓肿亚种和马赛亚种经Toll样受体2(Toll-like receptor 2,TLR2)介导的c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)诱导THP-1巨噬细胞内肿瘤坏死因子α(tumor necrosis factor α,TNF-α)和白细胞介素8(interleukin 8,IL-8)表达的相关分子机制,本研究将脓肿分枝杆菌脓肿亚种和马赛亚种感染THP-1巨噬细胞,细菌与巨噬细胞最佳感染之比为感染复数(multiplicity of infection,MOI)＝3,用荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测THP-1巨噬细胞感染两细菌亚种6 h后的胞内TNF-α和IL-8 mRNA水平,以及分别阻断TLR2、JNK 和ERK信号蛋白后TNF-α和IL-8 mRNA水平的变化。结果显示,脓肿分枝杆菌脓肿亚种和马赛亚种作用于THP-1巨噬细胞6 h后,均可诱导细胞内TNF-α和IL-8 mRNA水平显著上调,差异有统计学意义(P＜0.05);分别阻断TLR2、JNK和ERK信号蛋白,脓肿亚种感染THP-1巨噬细胞后胞内TNF-α和IL-8 mRNA上调水平出现明显抑制,差异有统计学意义(P＜0.05);分别阻断TLR2和JNK信号蛋白,马赛亚种感染THP-1巨噬细胞后胞内TNF-α和IL-8 mRNA上调水平均出现明显抑制,差异有统计学意义(P＜0.05);而阻断ERK信号蛋白后,马赛亚种组仅见IL-8 mRNA水平明显抑制,差异有统计学意义(P＜0.05),而TNF-α mRNA水平未见明显变化,差异无统计学意义(P＞0.05)。本研究提示,脓肿分枝杆菌脓肿亚种和马赛亚种均可作用于TLR2,诱导THP-1细胞内TNF-α和IL-8 mRNA水平上调,脓肿亚种可经JNK和ERK信号蛋白诱导TNF-α mRNA上调,马赛亚种可经JNK信号蛋白诱导TNF-α mRNA上调;脓肿亚种和马赛亚种诱导IL-8 mRNA上调可能与JNK和ERK信号蛋白相关。     

AiiA蛋白的可溶性表达及其抗菌活性研究

AHLs是革兰氏阴性细菌在增殖过程中产生的一类信号分子,与其致病性密切相关。AiiA蛋白作为一种胞内解酯酶．能水解致病菌产生的AHb分子,使内酯环开环后不能再激活某些胞外酶的表达．从而极大地减弱了细菌的致病性。本研究从苏云金芽孢杆菌LLB15中分离编码aiiA基因的质粒DNA。用PCR方法克隆面讲基因,并利用pET载体构建6-His融合表达质粒pET29a-aiiA,转化E.coli BL21（DE3）菌株,并筛选得到E.coli BL21（DE3）-pET29a-aiiA工程菌。在20℃的低温和0．8mmol／LIPTG条件下。经25h的诱导表达,获得了54．4ug／mL可溶性AiiA蛋白。通过镍柱亲和层析,在国内外首次纯化了带6-His标记的AiiA蛋白。水解活性和抗病性检测表明,该蛋白能水解AHLs分子．对胡萝卜欧文氏软腐病菌具有较强的抗病作用。     

Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor

Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K(m) (Mg·ATP) and V(max) values in Mg·ATP-dependent leukotriene-C4 transport. However, the V(max) values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.     

Requirement for Lamin B Receptor and Its Regulation by Importin β and Phosphorylation in Nuclear Envelope Assembly during Mitotic Exit

Lamin B receptor (LBR), a chromatin and lamin B-binding protein in the inner nuclear membrane, has been proposed to target the membrane precursor vesicles to chromatin mediated by importin β during the nuclear envelope (NE) assembly. However, the mechanisms for the binding of LBR with importin β and the membrane targeting by LBR in NE assembly remain largely unknown. In this report, we show that the amino acids (aa) 69–90 of LBR sequences are required to bind with importin β at aa 45–462, and the binding is essential for the NE membrane precursor vesicle targeting to the chromatin during the NE assembly at the end of mitosis. We also show that this binding is cell cycle-regulated and dependent on the phosphorylation of LBR Ser-71 by p34^cdc2 kinase. RNAi knockdown of LBR causes the NE assembly failure and abnormal chromatin decondensation of the daughter cell nuclei, leading to the daughter cell death at early G₁ phase by apoptosis. Perturbation of the interaction of LBR with importin β by deleting the LBR N-terminal spanning region or aa 69–73 also induces the NE assembly failure, the abnormal chromatin decondensation, and the daughter cell death. The first transmembrane domain of LBR promotes the NE production and expansion, because overexpressing this domain is sufficient to induce membrane overproduction of the NE. Thus, these results demonstrate that LBR targets the membrane precursor vesicles to chromatin by interacting with importin β in a LBR phosphorylation-dependent manner during the NE assembly at the end of mitosis and that the first transmembrane domain of LBR promotes the LBR-bearing membrane production and the NE expansion in interphase.     

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD⁺-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.     

青海藏区B型沙眼衣原体的分离培养与鉴定

【目的】对青海藏区沙眼患者标本进行沙眼衣原体分离培养与鉴定。【方法】分别采集患者的单眼结膜和结膜囊拭子标本至1 mL样本保护培养基中。取50μL样本采用离心法感染BGM细胞,37°C培养72 h,连续传代3次,相差显微镜观察衣原体包涵体。对临床样本和分离株分别进行主要外膜蛋白基因ompA序列分析。【结果】共采集了45例活动性沙眼患者的115份临床样本,其中54份样本为ompA PCR阳性,15份样本为沙眼衣原体培养阳性。ompA分析发现,青海藏区沙眼衣原体有3个不同的同源ompA变异株,均为基因B型,都包含有一个泌尿生殖道型沙眼衣原体特有密码子。分离株QH111L和QH111R分别来自编号111患者的左眼和右眼样本,它们ompA基因的可变区有一个非同义碱基差异。该碱基变异仅存在于111号患者的左眼样本中,说明QH111L可能是新出现的ompA突变体。【结论】青海藏区的眼型沙眼衣原体流行株为基因B型,至少存在3个不同的ompA变异株。从青海藏区分离培养了15株眼型沙眼衣原体,发现同一患者的左右眼样本中的沙眼衣原体有不同ompA。本研究为研制沙眼疫苗和诊断试剂奠定了基础,也将有助于理解沙眼的进化和传播。     

Molecular Cloning of Novel Cellulase Genes <Emphasis Type="Italic">cel</Emphasis>9A and <Emphasis Type="Italic">cel</Emphasis>12A from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> GXN151 and Synergism of Their Encoded Polypeptides

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.     

基于酵母水平的高通量药物筛选

随着后基因组时代的到来,越来越多的药物靶标蛋白将会被发现,基于靶标蛋白设计出的化合物也将大量涌现,高通量药物筛选日趋重要。酵母基因组的易操作性及其简单稳定的培养条件,使得该真核微生物成为一种理想的药物筛选工程细胞。本文讨论了选择酵母系统进行细胞水平筛选的优缺点,并从基于靶点和表型两种筛选模式对酵母水平的高通量药物筛选做一总结。