Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.     

兴化香葱降血脂作用的实验研究

研究了兴化香葱对高脂饲料喂养的鹌鹑血脂水平和肝脏脂肪化程度的影响。鹌鹑采食高脂饲料12天后,血清总胆固醇（TC）和总甘油三酯（TG）分别升高至原来的7倍和3倍。此后按500mg/（kg.d）的剂量饲以含兴化香葱干粉的高脂饲料40天,试验组动物的血清总胆固醇（TC）水平比高脂模型组降低了56.18%,效果非常显著（P〈0.01）,同时也显著低于阳性对照血脂康组（P〈0.01）。肝重系数较高脂模型组亦有明显下降（P〈0.05）,组织切片镜检可见肝脂肪变性程度减轻。以上结果提示,兴化香葱具有较强的降低血清胆固醇水平和减轻肝脏脂肪化程度的作用。     

Natural regulatory T cells mediate the development of cerebral malaria by modifying the pro-inflammatory response

Cerebral malaria (CM) is a severe neurologic complication that arises predominantly in children and non-immune adults infected with Plasmodium falciparum. In the current study, the dynamics of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and pro- and anti-inflammatory cytokines were analyzed in P. berghei ANKA (P.bANKA)-infected C57BL/6, BALB/c, and DBA/2 mice. We showed that C57BL/6 mice were susceptible to CM, while BALB/c and DBA/2 mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The proportion and absolute numbers of Tregs in BALB/c and DBA/2 mice were significantly higher than in C57BL/6 mice. The levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, IL-17 and NO in CM-susceptible C57BL/6 mice were obviously higher than in CM-resistant BALB/c and DBA/2 mice, while the level of the anti-inflammatory cytokine IL-10 was the opposite to that of pro-inflammatory cytokines, confirming that an appropriate balance between pro- and anti-inflammatory immune responses is essential to control the pathogenesis of severe malaria, and Tregs are important regulators if this balance is to be maintained. In vivo depletion of Tregs significantly protected C57BL/6 mice from experimental CM and the production of pro- and anti-inflammatory cytokines was reversed, indicating that this cell population contributes to pathogenesis by modulating the balance of pro- and anti-inflammatory responses. Our data demonstrate that Tregs mediate the incidence and outcome of CM in P.bANKA-infected mice by modifying the pro-inflammatory response.     

Perl语言环境下XML文档解析方法

数据准备是生物信息学研究中的重要步骤,数据准备过程中常常需要进行复杂的数据解析。本文详尽地描述了在Perl语言环境下如何利用XML::Smart模块对NCBI发布的同源基因数据集HomoloGene进行解析提取其中目标数据的方法及其技巧。对单个XML项目用对象实例-{‘项目名’}解析;对同层有多个项目的情况用对象数组解析。     

Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction. Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc-thiolate interaction. The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket. Here we establish the significance of each of these major features of the active site cleft by mutational analysis. Substitutions generally lead to substantially deleterious effects on K(m) and k(cat) parameters with respect to each of the cysteine, ATP, and tRNA(Cys) substrates. These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing. Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site.     

Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA

Transfer RNA can play a role in amino acid activation by aminoacyl-tRNA synthetases. For the prolyl-tRNA synthetase (ProRS) of Methanococcus jannaschii, which activates both proline and cysteine, the role of tRNA in amino acid selection and activation is of interest in the effort to understand the mechanism of the dual-specificity. While activation of proline does not require tRNA, whether or not tRNA is required in the activation of cysteine has been a matter of debate. Here, investigation of a series of buffer conditions shows that activation of cysteine occurs without tRNA in a wide-range of buffers. However, the extent of cysteine activation is strongly buffer-dependent, varying over a 180-fold range. In contrast, the extent of proline activation is much less sensitive to buffer conditions, varying over only a 36-fold range. We also find that addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual-specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis.     

小麦谷氨酸脱羧酶的纯化及部分性质研究

谷氨酸脱羧酶（ｇｌｕｔａｍａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）催化谷氨酸脱羧生成γ－氨基丁酸（γ－ａｍｉｎｏｂｕｔｙｒａｔｅ,ＢＡ）,植物中已从南瓜［１］、马铃薯和林生山黧豆［２］纯化了ＧＡＤ．ＧＡＤ活性在禾本科作物中作为...     

玉米根际联合固氮菌Klebsiella planticola 19—1固氮特性的研究

从新疆鄯善砍儿乡砂地生长的玉米根际分离到一株联合固氮菌Ｋ１９－１,经鉴定为植生克雷伯氏菌。Ｋ１９－１在４０℃,于无氮培养基上生长良好,并具有乙炔还原及＾１５Ｎ２还原能力。ｎｉｆＨｐｒｏｍｏｔｅｒ：ｌａｃＺ融合子在Ｋ１９－１中的表达在４０℃不受阻抑。ＮＨ４＾＋不抑制Ｋ１９－１的固氮酶活性,但阻抑ｎｉｆＨｐｒｏｍｏｔｅｒ：ｌａｃＺ融合子在Ｋ１９－１中表达。Ｋ１９－１在厌氧条件下具有固氮活性,在氧浓度增