H2O2诱导Neuro-2a细胞死亡机理的研究

细胞内线粒体呼吸链过程中的电子漏和神经细胞代谢的酶类如单胺氧化酶(MAO)等可产生活性氧物质(ROS)如H2O2等.ROS对细胞有毒性作用,导致细胞死亡,在许多疾病特别是神经退行性疾病中具有重要作用.我们用H2O2诱导N-2a神经母细胞瘤细胞,利用光镜、荧光显微镜、透射电镜观察了诱导的N-2a细胞的死亡,结果表明其死亡形式不同于典型的细胞凋亡,而类似于Ⅱ型神经细胞编程性死亡,死亡细胞染色质呈团块状凝集,细胞核膜仍保持完整.DNA不降解形成ladder,且不需要caspase-3,1的活性,但是H2O2诱导的Neuro-2a细胞死亡可以被Bcl-XL抑制.我们的结果可以说明,ROS介导的细胞毒性作用是导致Ⅱ型神经细胞编程性死亡的一个原因.     

慈竹BeSWEET4 2和BeSWEET1a 2基因的克隆和表达分析

糖外排转运蛋白(sugar will eventually be exported transporters, SWEET)在植物光合同化产物的运输中主要参与韧皮部糖的装载过程。该研究基于慈竹转录组库筛选出9条完整的BeSWEET序列,对其进行生物信息学分析,以慈竹茎和嫩叶的cDNA为模板对9条BeSWEET序列中的BeSWEET4 2和BeSWEET1a 2进行基因克隆,采用实时荧光定量(qRT PCR)分析其在叶肉、叶脉、根、茎中的表达水平以及外源糖诱导下的表达变化。结果表明：(1)系统进化分析显示,9条BeSWEET序列被分为4大类群,其中BeSWEET4 2与水稻SWEET4近缘,聚类到Clade Ⅱ,具有2个MtN3保守结构域;BeSWEET1a 2与水稻SWEET1a和SWEET1b近缘,聚类到Clade Ⅰ。(2)序列分析显示,慈竹BeSWEET4 2和BeSWEET1a 2分别编码256个和222个氨基酸,分别具有7个和5个跨膜结构域。(3)亚细胞定位预测显示：慈竹BeSWEET4 2和BeSWEET1a 2均定位于质膜。(4)qRT PCR结果显示,慈竹BeSWEET4 2和BeSWEET1a 2在叶肉、叶脉、根和茎中均有表达,分别在根和叶脉中最为显著,推测其可能协作参与了糖类从源到库的转运。(5)在外源己糖诱导下慈竹BeSWEET4 2和BeSWEET1a 2均显著上调表达,显示出对己糖的偏好性。该研究结果为进一步探讨慈竹BeSWEET蛋白调控糖转运的生物学功能奠定了基础。     

麻竹miR172a靶基因DlAP2的克隆及其表达

为了解开花麻竹(Dendrocalamus latiflorus)的Dl AP2基因功能,采用RT-PCR和RACE技术克隆了mi R172a靶基因AP2同源序列c DNA全长,命名为Dl AP2。结果表明,Dl AP2基因c DNA全长为1729 bp,包含5′端非编码区81 bp、开放阅读框1464 bp、3′端非编码区160 bp和24个碱基的Poly A尾巴,在编码框靠近3′端130 bp处有1个高度匹配mi R172a的结合位点(CTGCAGCATCATCAGGATTCT)。Dl AP2编码487个氨基酸的蛋白,具有两个AP2结构域,属于AP2/ERF家族AP2亚家族的AP2组,与来自其它单子叶植物的AP2蛋白均有较高同源性。RLM-5′RACE分析表明,mi R172a主要在靶序列的第11~12个碱基之间剪切靶基因Dl AP2的mi RNA。q RT-PCR结果表明,麻竹花芽中Dl AP2基因的表达规律与mi R172a表达变化正好相反,证明mi R172a对Dl AP2基因的表达具有调控作用。     

The effect of ellagic acid on caspase-3/bcl-2/Nrf-2/NF-kB/TNF-α /COX-2 gene expression product apoptosis pathway: a new approach for muscle damage therapy

Molecular Biology Reports - The goal of this study was to determine the protective role of ellagic acid (EA) against CCl4-induced muscle injury in rats. In this study, 36 Wistar albino rats...     

CCN2/decorin interactions: a novel approach to combating fibrosis?

CCN2 (connective tissue growth factor, CTGF), a member of the CCN family is overexpressed in fibrotic disease and is essential for the development of experimental fibrosis. Drugs targeting CCN2 action may therefore prove to be useful anti-fibrotic approaches. CCN2 acts via integrins and heparan sulfate-containing proteoglycans (HSPGs). In a recent study, Vial and colleagues (2011) show that decorin can bind CCN2. A peptide corresponding to the leucine rich repeats peptide 12 region of decorin can neutralize CCN2-mediated activity on C2C12 cells in vitro. Thus it is conceivable that this peptide could be used in the future as a novel antifibrotic approach.     

2′-p-Hydroxybenzoyl mussaenosidic acid,a new iridoid glucoside from Vitex negundo.

Ethanolic extract of the leaves of Vitex negundo yielded a new iridoid named 2′-p-hydroxybenzoyl mussaenosidic acid. The structure was established on the basis of chemical and spectral data.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Sensitivity to dietary obesity linked to a locus on Chromosome 15 in a CAST/Ei × C57BL/6J F2 intercross

Details of a new model of diet-dependent polygenic obesity are presented. CAST/Ei (Mus m. castaneus) mice remain lean after 12 weeks on a high-fat (32 kcal% fat) diet, while C57BL/6J mice become obese. The genes responsible for the obesity segregate in an F₂ population derived from an intercross between CAST/Ei and C57BL/6J mice. Quantitative trait analysis, with simple sequence length polymorphisms (SSLPs) at loci previously linked to rodent obesities, identified a quantitative trait locus (QTL) on Chromosome (Chr) 15, accounting for approximately 9% of the variance in adiposity and 14% of the variance in mesenteric depot size. This locus appears to be at the same location as the dietary obesity-3 (Do3) locus controlling body fat content, which was previously identified in an F₂ population derived from an SWR/J × AKR/J cross. This is also at the same location as the multigenic obesity-4 (Mob4) locus found in BSB mice, which display spontaneous polygenic obesity. Suggestive linkage also was found at loci close to the single gene mutations A ^y on Chr 2 and tub on Chr 7. Received 15 January 1996 / Accepted 12 May 1996     

Conformational studies on complexes of a diimine containing a (CH2)2 spacer: crystal structures of a double-stranded Zn(II) meso-helicate and an enantiopure Δ-Cu(II) monohelicate

Ni(II), Cu(II), Zn(II) and Cd(II) complexes of an N₄-donor Schiff base, containing (CH₂)₂ as spacer, have been prepared. The X-ray crystal structures of monohelical Ni(ETs) · H₂O and the homochirally crystallised Δ-Cu(ETs), as well as the meso-helicate Zn₂(ETs)₂ · MeCN [H₂ETs: N,N^′-bis(2-tosylaminobenzylidene)-1,2-diaminoethane] have been solved. In the latter, the ligand behaves as bis-bidentate, displaying a “C”-type arrangement, instead of the typical “S”-type fashion present in bis-helical dinuclear complexes.     

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2β interaction

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α^1–335 and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α^1–335. Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α^1–335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α^1–335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a K_D value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC₅₀ value of 92 μM.     

Does a light-harvesting protochlorophyllide a/b-binding protein complex exist?

Recent in vitro studies have led to speculation that a novel light-harvesting protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide oxidoreductases A and B containing nonphotoactive protochlorophyllide b and photoactive protochlorophyllide a, respectively. Functionally, LHPP has been hypothesized to play major roles in establishing the photosynthetic apparatus, in protecting against photo-oxidative damage during greening, and in determining etioplast inner membrane architecture. However, the LHPP model is not compatible with other studies of the pigments and the pigment-protein complexes of dark-grown angiosperms. Protochlorophyllide b, which is postulated to be the major light-harvesting pigment of LHPP, has, for example, never been detected in etiolated seedlings. This raises the question: does LHPP exist?     

On the Existence of a Possible A2A-D2-β-Arrestin2 Complex: A2A Agonist Modulation of D2 Agonist-Induced β-Arrestin2 Recruitment

Given that coactivation of adenosine A_2A (A_2AR) and dopamine D₂ (D₂R) receptors results in the coaggregation, cointernalization, and codesensitization of the A_2AR and D₂R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A_2AR agonist CGS21680 in A_2AR-D₂R-coexpressing cells to modulate the D₂R agonist-induced recruitment of β-arrestin2 to the D₂R by means of proximity-based bioluminescence resonance energy transfer (BRET²) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET² signal between β-arrestin2^RLuc and D_2LR^GFP2 upon D₂R activation, by increasing the potency of the D₂R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2^GFP2 and D_2LR^YFP as seen from the co-trafficking analysis. Furthermore, the A_2AR agonist advanced the time for the increase in Akt phosphorylation obtained with the D₂R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A_2AR-D₂R heteromers. Taken together, the results indicate that the antagonistic A_2AR-D₂R allosteric receptor-receptor interaction in A_2AR-D₂R heteromers favors β-arrestin2 recruitment to the D_2LR protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.     

小鼠Bcl2a1a蛋白的生物信息学预测分析

目的:分析小鼠Bcl2a1a全长基因的核苷酸序列及其编码蛋白的氨基酸序列,利用软件预测其蛋白的二、三级结构与特征。方法:运用生物信息学相关软件分析和预测人类BCL2A1和小鼠Bcl2a1a基因的同源区段,预测小鼠Bcl2a1a基因的启动子区域及蛋白跨膜区域与信号肽;利用软件模拟生成蛋白三级结构图像,并了解小鼠Bcl2a1a蛋白与其他蛋白的相互作用关系。结果:小鼠Bcl2a1a基因全长5497 bp,编码的蛋白含有172个氨基酸残基,相对分子质量为460 087.23,属于不稳定的疏水性蛋白。小鼠Bcl2a1a基因与人类BCL2A1基因的同源区域在≥200 bp的位置。2个软件预测的小鼠Bcl2a1a基因启动子最可能在3439～3543 bp和4600 bp,但由于没有预测到CpG岛存在,所以结果准确率较低。小鼠Bcl2a1a蛋白无跨膜结构域,无信号肽;在二级结构预测中,α螺旋为Bcl2a1a蛋白的主要折叠形式;该蛋白仅含有1个BCL结构域,且与Bbc3、Apaf1、Bcl2l2、Trp53、Bak1、Bid、Nfkb1、Jun、Rel、Rela等蛋白形成相互作用网络。结论:Bcl2a1a基因及蛋白的生物信息学分析为相关研究奠定了重要的信息基础。     

特应性皮炎患儿外周血miR-122a和miR-146a水平与Th1/Th2/Th17免疫平衡的相关性分析

摘要 目的：探讨特应性皮炎（AD）患儿外周血微小核糖核酸（miRNA）-122a和miR-146a水平与辅助性T细胞（Th）1/Th2/Th17免疫平衡的相关性。方法：选取2020年5月～2023年5月石家庄市妇幼保健院皮肤科收治的AD患儿100例为AD组,根据特应性皮炎评分（SCORAD）分为轻度组31例、中度组41例、重度组28例,另选取同期100名体检健康儿童为对照组。采用实时荧光定量聚合酶链式反应检测外周血miR-122a、miR-146a水平,流式细胞术检测外周血Th1、Th2、Th17细胞比例,酶联免疫吸附法检测外周血Th1、Th2、Th17相关细胞因子[白细胞介素（IL）-2、干扰素-γ（IFN-γ）、IL-4、IL-13、IL-17、IL-22]水平,并计算Th1/Th2/Th17比值。采用Pearson/Spearman相关性分析AD患儿外周血miR-122a、miR-146a与Th1、Th2、Th17和相关细胞因子及Th1/Th2/Th17的相关性。结果：AD组外周血miR-122a、miR-146a、Th1、Th1/Th2/Th17、IL-2、IFN-γ水平低于对照组,Th2、Th17、IL-4、IL-13、IL-17、IL-22水平高于对照组（P均＜0.05）。轻度组、中度组、重度组外周血miR-122a、miR-146a、Th1、Th1/Th2/Th17、IL-2、IFN-γ水平依次降低,Th2、Th17、IL-4、IL-13、IL-17、IL-22水平依次升高（P均＜0.05）。Pearson/Spearman相关性分析显示,AD患儿外周血miR-122a、miR-146a与Th1、Th1/Th2/Th17、IL-2、IFN-γ水平呈正相关（r/rs分别为0.679、0.677、0.684、0.706、0.693、0.689、0.671、0.694,P均＜0.001）,与Th2、Th17、IL-4、IL-13、IL-17、IL-22呈负相关（r/rs分别为-0.690、-0.680、-0.681、-0.669、-0.675、-0.676、-0.676、-0.686、-0.682、-0.674、-0.680、-0.689,P均＜0.001）。结论：AD患儿外周血miR-122a、miR-146a水平降低,与病情严重程度密切相关,可能通过调节Th1/Th2/Th17免疫平衡参与AD发生发展。