水稻花药总蛋白质双向电泳方法的改良与优化(简报)

双向电泳作为蛋白质组学核心技术之一,目前已广泛地应用在植物领域,并且成功应用于水稻代谢和调节等方面的研究。谢锦云等利用溶液法提取温敏核不育水稻花药总蛋白质,利用pH3-10线性胶条分离,经银染显色后检测到约1,000个     

Insights into the oligomeric states, conformational changes, and helicase activities of SV40 large tumor antigen

The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N- and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg(2+). The most C-terminal fragment (residues 628-708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N- and C-terminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.     

Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen

The large tumor antigen (LTag) of simian virus 40, an AAA(+) protein, is a hexameric helicase essential for viral DNA replication in eukaryotic cells. LTag functions as an efficient molecular machine powered by ATP binding and hydrolysis for origin DNA melting and replication fork unwinding. To understand how ATP binding and hydrolysis are coupled to conformational changes, we have determined high-resolution structures ( approximately 1.9 A) of LTag hexamers in distinct nucleotide binding states. The structural differences of LTag in various nucleotide states detail the molecular mechanisms of conformational changes triggered by ATP binding/hydrolysis and reveal a potential mechanism of concerted nucleotide binding and hydrolysis. During these conformational changes, the angles and orientations between domains of a monomer alter, creating an "iris"-like motion in the hexamer. Additionally, six unique beta hairpins on the channel surface move longitudinally along the central channel, possibly serving as a motor for pulling DNA into the LTag double hexamer for unwinding.     

雪衣藻Chlamydomonas nivalis适应温度周期性变化的生理响应和分子机制

【目的】雪衣藻(Chlamydomonas nivalis)分布于高山积雪和两极地区,可耐受低温胁迫和温度骤变,其适应温度变化的分子机制目前尚不清楚。【方法】本研究基于温度周期性变化下雪衣藻生理指标的响应规律,选择10个取样点进行转录组测序。运用加权基因共表达网络分析(weighted gene co-expression network analysis, WGCNA)划分得到17个共表达模块,从中找到5个与样品处理显著关联的模块,并对温度骤变的时间点进行基因差异表达分析。最后对筛选得到的基因集进行功能注释分析。【结果】转录组学分析显示,C. nivalis在温度周期变化下基因表达量发生了全局变化,其中果糖和甘露糖代谢通路、淀粉和蔗糖代谢通路、谷胱甘肽代谢通路以及抗坏血酸和醛酸代谢通路中关键酶的编码基因在低温下上调表达。研究还发现在温度周期变化下,C. nivalis中蛋白质质量控制系统、光合作用系统、DNA修复系统相关基因响应温度变化。【结论】本研究为揭示雪衣藻适应温度胁迫的分子机制提供了重要线索,丰富了生物抗逆基因资源库。     

Fatty acid and nutritive quality of chia (Salvia hispanica L.) seeds and plant during growth

The fatty acid (FA) profile, chemical composition, gross energy and organic matter digestibility of chia (Salvia hispanica L.) have been determined in the seed and in the plant collected at five progressive morphological stages from early vegetative to budding stage. The FA analyses disclosed quantitative differences between the plant stages that were characterised by a high percentage of polyunsaturated fatty acids (PUFA), which made up from 752 to 623 g/kg of the total FA of the plant during the growth cycle. The α-linolenic acid (ALA, C_18:3n–3) decreased from 649 g/kg, at the early vegetative stage, to 499 g/kg of the total FA, at the budding stage, while all the other FAs increased with increasing growth stage. The chia seed FAs were also highly unsaturated, with their main components being ALA (641 g/kg of the total FA) and linoleic acid (LA, C_18:2n–6; 188 g/kg of the total FA).The evolution of the quality of chia is closely related to the ageing of the plant. The chia plant provides a forage with a good nutritive value when harvested at a stage before the shooting period. After this, the nutritional quality of the plant considerably decreases with an increase in the fibrous fractions and a dramatical decrease of the crude protein content.     

Design and synthesis of AX7574: a microcystin-derived, fluorescent probe for serine/threonine phosphatases

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.     

冠状动脉内放射抑制球囊扩张术后ERK1/2活性及c-fos基因表达

本文旨在观察血管内放射对冠状动脉球囊扩张术后细胞外信号调节激酶１／２（ＥＲＫ１／２）及ｃ－ｆｏｓ基因表达的影响。实验对猪的左冠状动脉前降支或回旋支行球囊行过度扩张术,术后即刻能过血管内放射治疗系统对猪冠状动脉损伤局部给予２０Ｇｙ的放射剂量,分别是在术后３ｄ和３０ｄ处死动物,留取目标血管组织。通过反转录－聚合酶链反应定量检测血管内入射对球囊扩张术后血管组织ｃ－ｆｏｓ ｍＲＮＡ的表达,采用生化方法测定     

Phosphorylated IkappaBalpha is a component of Lewy body of Parkinson's disease

Ubiquitin is one of the major components of Lewy bodies (LB), the pathological hallmark of Parkinson's disease (PD). Here, we identified that a phosphorylated form of IkappaBalpha (pIkappaBalpha), an inhibitor of NF-kappaB, and SCF(beta-TrCP), the ubiquitin ligase of pIkappaBalpha, are components of LB in brains of PD patients. In vitro studies identified those proteins in the ubiquitin- and alpha-synuclein (known as the major component of LB)-positive LB-like inclusions generated in dopaminergic SH-SY5Y cells treated with MG132, a proteasome inhibitor. Intriguingly, IkappaBalpha migration into such ubiquitinated inclusions in cells treated with MG132 was inhibited by a cell-permeable peptide known to block phosphorylation of IkappaBalpha, although this peptide did not influence cell viability under proteasomal inhibition. Our results indicate that phosphorylation of IkappaBalpha plays a role in the formation of IkappaBalpha-containing inclusions caused by proteasomal dysfunction, and that the generation of such inclusion is independent of cell death caused by impairment of proteasome.