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。     

SIRT1/PGC-1: a neuroprotective axis?

Neurodegenerative diseases are more and more prevalent in our aging societies. A rapid overview of the etiology of many neurodegenerative diseases like Alzheimer, Parkinson, Huntington disease and amyotrophic lateral sclerosis suggests a tight link with mitochondrial dysfunction. Since it has been recently demonstrated that activation of the SIRT1/PGC-1 pathway, in a metabolic context promotes mitochondrial function, we performed a detailed literature review on the implication of this pathway in neurodegeneration. Interestingly, transgenic mice with impaired PGC-1 expression have neurodegenerative lesions and show behavioural abnormalities. As evidenced from independent investigations, enhanced SIRT1 activity has been demonstrated to protect against axonal degeneration and to decrease the accumulation of amyloid beta peptides, the hallmark of Alzheimer disease, in cultured murine embryonic neurons. In addition, several studies suggest that resveratrol, a specific activator of SIRT1, could have protective effects in animal models of neurodegenerative diseases. Taken together, these results strongly suggest that the modulation of the SIRT1/PGC-1 pathway, which has not been well documented in the central nervous system, could become the cornerstone for new therapeutical approaches to combat neurodegeneration.     

Is PECAM-1 a mechanoresponsive molecule?

Endothelial cells are capable of responding to fluid shear stress, but the molecular mechanism for this biological response remains largely unknown. Our studies indicate that the cell-cell adhesion site is a possible site of flow sensing. PECAM-1, a cell adhesion molecule localized to the interendothelial cell adhesion site, is tyrosine-phosphorylated when endothelial cells are exposed to physiological levels of fluid shear stress. This PE-CAM-1 phosphorylation initiates a signaling cascade leading to ERK activation. Here we review what is known about PECAM-1 tyrosine phosphorylation and suggest a possible role of PECAM-1 in mechanosensing by endothelial cells.     

Possible trisomy 1q25→1q32 in a malformed girl with a de novo insertion in 1q

Summary A newborn female is described who exhibited a characteristic facial dysmorphology including deep-set eyes, broad nasal bridge, small mouth, higharched and narrow palate, severely receding mandible and misshapen ears; constant flexion of the proximal interphalangeal joints, and short distal phalanges and nails of fingers; a congenital heart defect; marked muscular hypotonia, motor and growth retardation. She died at 4 months of age. Her karyotype revealed an additional band in 1q. Banding patterns and clinical picture suggest duplication of the segment 1q251q32.     

Caspase-1 and IL-1β Processing in a Teleost Fish

Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β.     

Is 1-nitro-1-triazene a high energy density material?

An azo bridge (–N?=?N–) can not only desensitize explosives but also dramatically increase their heats of formation and explosive properties. Amino and nitro are two important high energy density functional groups. Here, we present calculations on 1-nitro-1-triazene (NH₂–N?=?N–NO₂). Thermal stability and detonation parameters were predicted theoretically at CCSD(T)/6-311G* level, based on the geometries optimized at MP2/6-311G* level. It was found that the p?→?π conjugation interaction and the intramolecular hydrogen bonding that exist in the system together increase the thermal stability of the molecule. Moreover, the detonation parameters were evaluated to be better than those of the famous HMX and RDX. Finally, the compound was demonstrated to be a high energy density material.     

拟南芥DBB 1 a（double B-box 1a）蛋白的表达、纯化及Western blotting检测

PCR扩增拟南芥（Arabidopsis thaliana）DBB1a cDNA的保守区段（GenBank登录号：AT2G21320）,转化到冷诱导表达载体pCold TF上,构建pCold-DBB1a重组质粒,转化大肠杆菌DH5a.15℃下IPTG诱导表达融合蛋白,并通过SDS-PAGE检测.证实目的蛋白以可溶形式在约20 kD处高效表达,与预期蛋白大小相吻合.表达蛋白经Ni琼脂糖凝胶亲和层析纯化,SDS-PAGE及Western blotting检测证实纯化后获得高纯度融合蛋白,这为进一步研究DBB1a功能奠定了基础.     

Type 1 diabetes as a relapsing-remitting disease?

Chronic immunological processes that underlie persistent viral infections and autoimmune disorders such as multiple sclerosis can be relapsing-remitting in nature. The progressive loss of beta-cell mass during the development of autoimmune type 1 diabetes (T1D) can also be non-linear, but the exact nature and kinetics of the immunological processes that govern T1D are not known. Here, we propose that the immunological process that is at the root of T1D is relapsing-remitting in nature and discuss the unresolved controversies and therapeutic implications of this hypothesis.     

Does Rap1 deserve a bad Rap?

The Ras superfamily of small G proteins is remarkable for both its diversity and physiological functions. One member, Rap1, has been implicated in a particularly wide range of biological processes, from cell proliferation and differentiation to cell adhesion. But the diversity of Rap1 has lead to contradictory reports of its effects. Originally identified as an antagonist of Ras-induced transformation, Rap1 can oppose other actions of Ras including regulation of cell growth and differentiation, integrin-dependent responses and synaptic plasticity. Furthermore, recent evidence confirms that Rap1, like Ras, can activate the MAP kinase cascade (ERK) in several cell types. These diverse functions of Rap1 underscore that the activation and action of Rap1 are regulated by complex factors that are cell-type specific.     

斑马鱼sat1.a突变体的鉴定

目的在我们的前期研究工作中,通过Tol2转座子介导的插入突变,筛选到了一批组织特异性表达绿色荧光蛋白GFP的斑马鱼品系。其中一个品系Tol2:20141221t的GFP在神经系统中表达,但还没有鉴定到Tol2转座子插入到基因组什么位置,造成了哪个基因的突变。本文的主要研究目标就是对这一由Tol2转座子插入诱导的斑马鱼突变品系进行鉴定。方法使用交错式热不对称PCR(thermal asymmetric interlaced PCR,TAIL-PCR)鉴定Tol2转座子插入的基因组位置;利用原位杂交检测被突变的基因的时空表达是否与该品系GFP表达具有一致性;筛选鉴定纯合体突变体,并进一步分析该基因突变造成的发育缺陷。结果在该品系中,Tol2转座子插入到了亚精胺/精胺N1-乙酰基转移酶1a(spermidine/spermine N1-acetyltransferase 1a,sat1.a)的第八个内含子区域,致使sat1.a基因转录提前终止。我们筛选到了sat1.a纯合突变体,但是没有检测到明显的发育缺陷。结论筛选鉴定的Tol2转座子介导的斑马鱼sat1.a突变体没有明显的发育缺陷,但可以作为研究神经系统发育的有力工具。     

胸腺素a1与复合a干扰素融合蛋白的表达及其生物学活性

实验旨在获得具有双重生物学活性的重组胸腺素a1(Thymosin alpha1, TM-a1)与复合a干扰素(IFNa-con)融合蛋白。选择大肠杆菌偏爱的密码子, 将合成的TM-a1与IFNa-con编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b(+)、在宿主菌BL21(DE3)-Codon plus-RP-X中成功表达了可溶性融合蛋白(TM-a1-IFN-con)。表达量占总蛋白的20%以上。通过硫酸铵沉淀、疏水层析、阴离子交换层析、阳离子交换层析、分子筛层析后, 产品纯度达到96%以上。采用细胞病变抑制法测定融合蛋白的抗病毒活性, 采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响。结果表明, 融合蛋白的抗病毒活性优于市售的IFNa1b和IFNa2a。对小鼠脾淋巴细胞增殖的影响与市售的合成胸腺素a1相同。已有研究证实, 该融合蛋白具有良好的体外抗HBV作用, 其体外抗HBV活性比联合应用TM-a1和干扰素a强, 且细胞毒性明显低于联合应用TM-a1和干扰素a。以上结果表明, 通过大肠杆菌表达的可溶性融合蛋白(TM-a1- IFN-con), 既具有良好的干扰素a抗病毒作用, 也具有胸腺素a1促淋巴细胞增殖作用。     

小鼠胚胎干细胞p16^INK4a基因外显子1a打靶研究

在活体水平上,小鼠p16^INK4a基因是否具有抑制肿瘤发生和发展的功能是一个悬而未决的问题。利用筛选基因组文库得到的小鼠p16^INK4a基因组DNA片段,构建了针对小鼠p16^INK4a基因外显子1a的基因打靶载体,其短臂为1.5kbEco81 I/AccⅡ片段,长壁为5.9kb Xba I/Xho I片段。打靶载体经线性化和纯化后通过电穿孔转导小鼠R1 ES细胞,获得37个G418和Gancyclovir双药抗性克隆。用Southern杂交法对双药抗性克隆进行鉴定,获得一个敲除了p16^INK4a基因外显子1a的阳性ES细胞克隆。     

Polydom/SVEP1 is a ligand for integrin α9β1

A variety of proteins, including tenascin-C and osteopontin, have been identified as ligands for integrin α9β1. However, their affinities for integrin α9β1 are apparently much lower than those of other integrins (e.g. α3β1, α5β1, and α8β1) for their specific ligands, leaving the possibility that physiological ligands for integrin α9β1 still remain unidentified. In this study, we found that polydom (also named SVEP1) mediates cell adhesion in an integrin α9β1-dependent manner and binds directly to recombinant integrin α9β1 with an affinity that far exceeds those of the known ligands. Using a series of recombinant polydom proteins with N-terminal deletions, we mapped the integrin-binding site to the 21st complement control protein domain. Alanine-scanning mutagenesis revealed that the EDDMMEVPY sequence (amino acids 2636-2644) in the 21st complement control protein domain was involved in the binding to integrin α9β1 and that Glu(2641) was the critical acidic residue for the integrin binding. The importance of this sequence was further confirmed by integrin binding inhibition assays using synthetic peptides. Immunohistochemical analyses of mouse embryonic tissues showed that polydom colocalized with integrin α9 in the stomach, intestine, and other organs. Furthermore, in situ integrin α9β1 binding assays using frozen mouse tissues showed that polydom accounts for most, but not all, of the integrin α9β1 ligands in tissues. Taken together, the present findings indicate that polydom is a hitherto unknown ligand for integrin α9β1 that functions as a physiological ligand in vivo.