瓜子金皂甙丙和丁的化学结构研究

自草药瓜子金(Polygala japonica Houtt)地上部分又分离得到两种皂甙,即瓜子金皂甙丙和皂甙丁,它们的分子式均为 C_(42)H_(68)O_(15),为同分异构物。两者酸水解均得瓜子金皂甙元和 D-葡萄糖,但碱水解时,只有皂甙丁可被水解,产物和酸水解相同。经~1HNMR,~(13)CNMR,MS 推定它们的结构式,瓜子金皂甙丙为3-O-[β-D-葡萄吡喃糖(1→2)-β-D-葡萄吡喃糖]瓜子金皂甙元;而瓜子金皂甙丁为28-O-[β-D-葡萄吡喃糖(1→2)-β-D-葡萄吡喃糖]瓜子金皂甙元。上述两种皂甙均为新化合物。     

不同植物中推定蓖麻烯合酶基因的生物信息学分析

利用Gen Bank中已登录的完整的麻风树、乳浆大戟、蓖麻和乌桕中的13个蓖麻烯合酶(Casbene synthase,CS;EC 4.6.1.7)基因序列,通过生物信息学方法对其核酸及氨基酸序列、组成成分、导肽、信号肽、跨膜结构域、疏水性/亲水性、蛋白质的二级结构、三级结构及功能域等进行了分析预测。结果表明,13个CS基因的ORF长度均在1 647~1 845 bp,蛋白分子量均在63.0~70.8 k D,终止密码子为TGA或TAA,理论等电点均小于7.0,表明CS蛋白呈酸性。氨基酸含量最高的均为亮氨酸。核苷酸同源性比较分析表明,CS基因主要分为两类。导肽预测发现其中6个CS具有导肽,均为叶绿体导肽。信号肽和扩模结构域预测发现这些CS不存在信号肽和跨膜结构域,肽链整体呈现为亲水性。这些CS的主要二级结构元件为α-螺旋,并且都包含两个萜类合酶功能域。以上研究为进一步探索CS基因的功能提供一定理论依据。     

慢病毒载体介导IκB激酶-α(IKKα)基因RNA干扰转基因小鼠的制备

IκB激酶-α(IKKα)的功能与淋巴形成和乳腺发育相关,在乳腺肿瘤中观察到IKKα蛋白的过量表达.应用小鼠受精卵卵周隙注射慢病毒载体的方式,建立IKKα基因RNAi转基因小鼠模型,为在体内研究IKKα基因的生物学功能及其与肿瘤发生的关系创造条件.实验构建了针对IKKα基因RNAi(剔降)的慢病毒载体,并将载体导入小鼠受精卵卵周隙,获得携带该载体的转基因小鼠模型,经PCR鉴定首建鼠阳性率为15%.转基因小鼠外周血细胞IKKαmRNA表达量明显降低.初步表型观察分析,IKKα基因RNAi小鼠发育正常,进一步的分析工作正在进行中.     

Effects of Arachidonic Acid on the Lysosomal Ion Permeability and Osmotic Stability

In this study, we investigated the effects of arachidonic acid, a PLA₂-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K⁺/H⁺ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.     

A Protein Encoded by din1, a Dark-Inducible and Senescence-Associated Gene of Radish, Can Be Imported by Isolated Chloroplasts and Has Sequence Similarity to Sulfide Dehydrogenase and Other Small Stress Proteins

In an attempt to isolate cDNA clones for dark-inducible chloroplastproteins, we screened a cDNA library which was prepared fromradish cotyledons by a two-step method. The source plants weregrown under continuous light for 14 d and kept in darkness for24 h. One of the selected clones, S2D12, corresponded to thedin1 gene which we previously reported as a dark-inducible,senescence-associated gene [Azumi and Watanabe (1991) PlantPhysiol. 95: 577]. A 22 kDa polypeptide was produced from thecDNA in an in vitro expression system in the presence of [³⁵S]methionine.This polypeptide was capable of being imported by isolated chloroplasts,processed to a smaller mature form and localized in the stromalfraction. As the amino acid sequence of the putative matureprotein has no homology to any known chloroplast protein, din1was suggested to be the first gene for a chloroplast proteinwhich is negatively controlled by light. The putative matureprotein has similarity to sulfide dehydrogenase from Wolinellasuccinogenes and other small stress proteins; glpE and pspEfrom Escherichia coli and hsp67B2 from Drosophila melanogaster. ¹ The nucleotide sequence data in this paper has been submittedto EMBL, GenBank and DDBJ Data Libraries under the acces sionnumber AB004242 ² Present address: The Institute of Physical and Chemical Research(RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-01 Japan     

Adiponectin 基因剔除LacZ基因敲入小鼠模型的建立

adiponectin是脂肪细胞特异分泌的一种活性蛋白质,具有增加胰岛素敏感性、抗炎及抗动脉硬化等活性.建立adiponectin基因剔除β-半乳糖苷酶基因(LacZ)敲入小鼠模型,可为整体动物水平研究adiponectin基因功能及其表达调控机制等提供理想工具.根据生物信息学方法获得adiponectin基因组序列,设计基因剔除及敲入策略,在adiponectin基因第2和第3号外显子剔除的同时,在其ATG和信号肽序列后顺接LacZ基因完整编码序列,构建完成了Adipo-LacZ-XpPNT基因剔除质粒.通过电穿孔将打靶质粒转入ES细胞,以G418和ganciclovir进行药物筛选,获得药物抗性的ES细胞克隆,PCR和DNA印迹鉴定出正确同源重组克隆.将同源重组的ES细胞克隆注入小鼠囊胚得到嵌合体小鼠,嵌合体小鼠与C57BL/6J小鼠交配产生杂合子小鼠,杂合子间交配获得adiponectin基因剔除LacZ基因敲入纯合子小鼠.经RT-PCR、RNA印迹和ELISA检测证实纯合子小鼠脂肪和血清中adiponectin基因表达呈阴性.RT-PCR、RNA印迹及蛋白质印迹检测发现,LacZ基因在突变小鼠脂肪组织中有特异性表达,其表达谱与内源性adiponectin基因的表达谱一致.但在脂肪组织及外周血中未能检测到LacZ活性,且血清中LacZ蛋白亦呈阴性.由此成功建立了adiponectin基因完全灭活及LacZ基因以内源性adiponectin基因表达谱表达的小鼠模型,为进一步研究该基因功能及其表达调控创造了有利条件.     

Relationship between raman spectroscopic lines and growth ofRhizobium japonicum

The Raman spectroscopic lines of liquid cultures ofRhizobium japonicum have been compared with electron microscopic examinations and growth measurements of these cells. The results showed that the significant Raman lines are related to the reproduction activities of the procaryotic cells.     

Mechanism of cytosol phospholipase C and sphingomyelinase-induced lysosome destabilization

Lysosomal disintegration may cause apoptosis, necrosis and some diseases. However, mechanisms for these events are still unclear. In this study, we measured lysosomal beta-hexosaminidase free activity, membrane potential and intralysosomal pH. The results revealed that the cytosolic extracts of rat hepatocytes could increase the lysosomal permeability to both potassium ions and protons, and osmotically destabilize lysosomes via K(+)/H(+) exchange. The effects of cytosol on lysosomes could be completely abolished by D609, which inhibited both phospholipase C and sphingomyelinase, and partly prevented by sphingomyelinase inhibitor Ara-AMP, but not by the inhibitors of PLA(2). Moreover, purified phospholipase C could destabilize the lysosomes while phospholipase A(2) and phospholipase D did not produce such effects. The cytosolic phospholipases hydrolyzed lysosomal membrane phospholipids by 50%, which could be prevented by D609. Disintegration of the cytosol-treated lysosomes biphasically depended on the cytosolic [Ca(2+)]. The cytosol did not disintegrate lysosomes below 100 nM or above 10 muM cytosolic [Ca(2+)], but markedly destabilized lysosomes at about 340 nM [Ca(2+)]. The results suggest that cytosolic phospholipase C and sphingomyelinase may be responsible for the alterations in lysosomal stability by increasing the ion permeability.     

The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus

In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.