Upregulation of miR-23a～27a～24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a∼27a∼24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a∼27a∼24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a∼27a∼24-2 cluster provides new insights in the development of novel therapeutics for cancer.     

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。     

H_2O_2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H_2O_2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H_2o_2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H_2O_2诱导的Neuro-2a细胞死亡可以被Bcl-X_L,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。     

Epac2: a sulfonylurea receptor?

Sulfonylureas are widely used oral drugs in the treatment of diabetes mellitus. They function by the inhibition of ATP-sensitive K+ channels in pancreatic β-cells, which are thus considered the 'classical' sulfonylurea receptor. Next to the ATP-sensitive K+ channels, additional sulfonylurea-interacting proteins were identified, which might contribute to the physiological effects of this drug family. Most recently, Epac2 (exchange protein directly activated by cAMP 2) was added to the list of sulfonylurea receptors. However, this finding caused controversy in the literature. The critical discussion of the present paper comes to the conclusion that sulfonylureas are not able to activate Epac2 directly and are unlikely to bind to Epac2. Increased blood glucose levels after food intake result in the secretion of insulin from pancreatic β-cells. Glucose levels are detected 'indirectly' by β-cells: owing to increased glycolysis rates, the ratio of cellular ATP/ADP increases and causes the closure of ATP-sensitive K+ channels. In consequence, cells depolarize and voltage-dependent Ca2+ channels open to cause an increase in the cellular Ca2+ concentration. Finally, Ca2+ induces the fusion of insulin-containing granules with the plasma membrane. Sulfonylureas, such as tolbutamide, glibenclamide or acetohexamide, form a class of orally applicable drugs used in the treatment of non-insulin-dependent diabetes mellitus.     

H2O2诱导Neuro—2a细胞死亡机理的研究

Reactive oxygen species (ROS), such as H2O2, can be produced by enzymes involved in electron leakage of respiration chain in mitochondria, and by neurochemical enzymes such as monoamine oxidase in neural cells. ROS are toxic to cells, and can result in cell death. ROS also play an important role in some diseases, especially in neurodegenerative diseases by yet unknown mechanisms. In the current research, the N-2a neuroblastoma cell was treated with H2O2, and the morphological changes of cell death were characterized. Our results show that N-2a cell death is different from classical apoptosis, but belongs type II nerve cell programmed death, which shows condensed chromatin within intact nuclear envelope and no apoptotic body. The chromatin DNA of dead cells shows no internucleosomal cleavage, as well as no requirement for caspase-3, 1 activity. However, the H2O2-induced N-2a cell death can be inhibited by Bcl-XL. It can be concluded that type II nerve cell death is the result of cell toxicity mediated by ROS. The results pave the way for further research of type II nerve cell death.     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

转GmTMT2a基因玉米抗旱功能分析

生育酚具有很强的抗氧化功能,其中α-生育酚是最有效的组分。研究了α-生育酚含量提高的转GmTMT2a基因植株（TP）和野生型植株（WT）在干旱条件下的响应差异。结果表明,TP植株和WT植株中H2O2 含量均有所增加,但TP植株中累积了更少的H2O2;抗氧化酶类SOD、POD和CAT的酶活测定结果表明,CAT酶活性在TP植株中的增幅最大;抗旱相关基因表达分析结果显示,P5CS和TPS在TP植株中的表达显著上调。推测转GmTMT2a基因后,提高了CAT的酶活以及P5CS和TPS的表达量,进而增强了植株的抗旱性。     

应用Ad Easy腺病毒载体系统构建人肌浆网钙离子ATP酶2a(SERCA2a)

目的:利用Ad easy腺病毒表达系统构建含人肌浆网钙离子ATP酶2a(SERCA2a)基因重组腺病毒,并在HEK293细胞中扩增制备重组腺病毒.方法:将人SERCA2a基因全长c DNA(3700bp)插入到腺病毒穿梭载体pAdTrack-CMV,成功构建pAd-TrackCMV SERCA2a重组质粒,经Pme I酶切线性化,采用电击法转入到已含Ad easy质粒的电感受态菌BJ5183进行重组.挑选同源重组质粒,Pac I酶切线性化转染HEK293细胞包装成重组腺病毒颗粒,荧光检测有绿色荧光蛋白表达.将重组病毒和SD大鼠心肌细胞共培养,western-blot检测SERCA2a可以在大鼠心肌细胞过表达且影响了胞内SERCA2a的活性.结果:成功包装含人SERCA2a基因的重组腺病毒,并可以有效感染SD大鼠心肌细胞.结论:利用新型腺病毒载体在短时间内成功构建了携带有人SERCA2a基因的腺病毒,为以后进一步研究人SERCA2a基因治疗提供了新途径.     

Prolyl hydroxylase 2: a novel regulator of β2 -adrenoceptor internalization

Adrenergic receptor (AR)-mediated signalling is modulated by oxygen levels. Prolyl hydroxylases (PHDs) are crucial for intracellular oxygen sensing and organism survival. However, it remains to be clarified whether or how PHDs are involved in the regulation of β(2) -adrenoceptor (β(2) -AR) signalling. Here we show that PHD2 can modulate the rate of β(2) -AR internalization through interactions with β-arrestin 2. PHD2 hydroxylates β-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of β-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with β(2) -AR into the cytosol. β(2) -AR internalization is critical to control the temporal and spatial aspects of β(2) -AR signalling. Identifying novel regulators of β(2) -AR internalization will enable us to develop new strategies to manipulate receptor signalling and provide potential targets for drug development in the prevention and treatment of diseases associated with β(2) -AR signalling dysregulation.     

长沙市2～12岁哮喘儿童睡眠紊乱研究

目的:对长沙市2～12岁非哮喘儿童及哮喘儿童睡眠状况进行调查,比较常见睡眠障碍在两组中的发生率区别.方法:于2006年6月至10月对长沙市五个区的小学及幼儿园采取整群随机抽样方法进行调查抽取2～12岁健康儿童3420例及湘雅二院儿童哮喘专科门诊随机抽取2～12岁符合哮喘诊断标准[6]的哮喘儿童248名进行问卷调查,由专业人员指导家长填写.结果:哮喘儿童睡眠障碍发生率58%,显著高于健康儿童组38%水平.哮喘儿童中,每周1～3次以上打鼾的32例(12.9%),喉头哽咽的5例(2%),呼吸暂停的3例(1.2%),辗转不安的36例(14.5%),用口呼吸30例(12.1%),多汗97例(39.1%),肢体抽动18例(7.3%),磨牙34例(13.7%),说梦话12例(4.8%),梦游2例(0.8%),夜间尿床11例(4.4%),觉醒憋醒8例(3.2%),夜间清醒6例(2.4%),入睡过早5例(2%),易惊醒10例(4%),尖叫哭喊6例(2.4%).其中,每周1～3次以上打鼾、辗转不安、用口呼吸、多汗、肢体抽动、觉醒憋醒、易惊醒七项存在显著性差异(P＜0.05).结论:哮喘儿童睡眠障碍发生率显著高于健康儿童,应加强对哮喘儿童睡眠障碍的干预.     

PmST2: a novel Pasteurella multocida glycolipid α2-3-sialyltransferase

Pasteurella multocida (Pm) is a multi-species pathogen that causes diseases in animals and humans. Sialyltransferase activity has been detected in multiple Pm strains and sialylation has been shown to be important for the pathogenesis of Pm. Three putative sialyltransferase genes have been identified in Pm genomic strain Pm70. We have reported previously that a Pm0188 gene homolog in Pm strain P-1059 (ATCC 15742) encodes a multifunctional sialyltransferase (PmST1). We demonstrate here that while PmST1 prefers to use oligosaccharides as acceptors, PmST2 encoded by the Pm0508 gene homolog in the same Pm strain is a novel glycolipid α2-3-sialyltransferase that prefers to use lactosyl lipids as acceptor substrates. PmST2 and PmST1 thus complement each other for an efficient synthesis of α2-3-linked sialosides with or without lipid portion. In addition, β1-4-linked galactosyl lipids are better PmST2 substrates than β1-3-linked galactosyl lipids. PmST2 has been used successfully in the preparative scale synthesis of sialyllactosyl sphingosine (lyso-GM3), which is an important glycolipid and an intermediate for synthesizing more complex glycolipids such as gangliosides.     

猪血浆a2巨球蛋白部分性质的研究

对猪血浆α２巨球蛋白性质研究表明其大部分理化性质与人α２巨球蛋白十分相似,而且猪α２巨球蛋白也存在有活性的天然的Ｓｌｏｗ－Ｆｏｒｍ和构象发生变化并不可逆失活的Ｆａｓｔ－Ｆｏｒｍ。猪α２巨球蛋白经测定其ＰＩ为４．５５,５．１。紫外吸收光谱在２７６ｍｍ处有最大吸收,其糖含量为９．３％。氨基酸组成表明酸性氨基酸含量较高。圆二色谱分析说明分子中含有较多的Ｂ折叠。它还具有连接锌离子的性质。     

Tolypocladin — a new metal-chelating 2-aza-anthraquinone fromTolypocladium inflatum

Summary A new chelator of di- and trivalent cations (tolypocladin) was isolated from the mycelium ofTolypocladium inflatum DSM 915. The structure has been determined by NMR methods as 3-methyl-5,6(7),8-trihydroxy-2-aza-anthraquinone. The ultraviolet, visible and fluorescence spectral properties of some metal complexes (in methanol) are described. The compound forms water-soluble fluorescent aluminium complexes. Its production is dependent on zinc ions in the medium. It serves as an endogenous hydrogen acceptor under oxygen limitation in the producing strain,T. inflatum.     

Hb a 2 gluß2 (Hb I) in a Caucasian family: Independent mutation or common origin?

Summary Hemoglobin 16glu (Hb I-Skamania) was confirmed in six persons of a Caucasian family by amino acid analysis of the abnormal tryptic peptide, T3,4. The confirmation of Hb 16glu in Caucasians and the apparent absence of Hb I in racially unmixed Negroes or in American Indians suggest that Hb 16gly may be of European origin. However, an independent European and African origin can currently not be ruled out. The origin of seven other rare hemoglobin mutants is also uncertain. Independent genetic origin of a rare mutant is confirmed only when a new mutation can be proved.     

LTBP-2 acts as a novel marker in human heart failure – a preliminary study

Background: We have observed increased expression of latent TGF-β binding protein (LTBP)-2 mRNA in human failing hearts. This study was aimed to further confirm LTBP-2 act as a novel marker in human acute heart failure.

Methods and results: We demonstrated that median level of LTBP-2 in myocardial samples from heart failure patients was significantly elevated, and TGF-β1 significantly promoted LTBP-2 expression in neonatal rat cardiomyocytes. To investigate the potential of LTBP-2 as a biomarker to diagnose heart failure with reduced ejection fraction (HFREF), another cohort of 133 consecutive patients with dyspnea were enrolled. In receiver operating characteristic (ROC) curve analyses to detect HFREF, LTBP-2 achieved an area under curve (AUC) of 0.67 (95% confidence intervals (CI) 0.58–0.75), comparable to the diagnostic ability of NT-proBNP 0.68 (95% CI 0.59–0.77).

Conclusion: The serum LTBP-2 levels might act as a promising biomarker in HFREF.