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.     

α-Synuclein Immunoisolation of Glial Inclusions from Multiple System Atrophy Brain Tissue Reveals Multiprotein Components

Immunohistochemical studies have shown that oligodendroglial inclusions in multiple system atrophy contain alpha-synuclein, a synaptic protein also found in Lewy bodies in Parkinson's disease. We have now used density gradient enrichment and an anti-alpha-synuclein immunomagnetic technique to isolate pure and morphologically intact oligodendroglial inclusions from brain white matter of patients dying with multiple system atrophy. Filamentous inclusion structures were obtained only from multiple system atrophy tissue, but not from normal brain tissues, or from multiple system atrophy tissue processed without anti-alpha-synuclein antibody. We confirmed the purity and morphology of isolated inclusions by electron microscopy. The inclusions comprised multiple protein bands after separation by polyacrylamide gel electrophoresis. Immunoblotting demonstrated that these proteins included alpha-synuclein, alphaB-crystallin, tubulins, ubiquitin, and prominent, possibly truncated alpha-synuclein species as high-molecular-weight aggregates. Our study provides the first biochemical evidence that oligodendroglial inclusion filaments consist of multiple protein components, suggesting that these inclusions may form as a result of multiprotein interactions with alpha-synuclein.     

阳原石核的动态类型学研究及其工艺思想分析

本文从动态类型学角度把阳原石核的工艺程序划分为四道工序和两个亚类型,论证了这种石核发生形态变化的机制。在工艺特征上,阳原石核与日本北海道的(山上下)下型细石核相当,从而证明两地在旧石器时代晚期曾有共同的工艺思想。     

Establishment of Hairy Root Cultures by Agrobacterium Rhizogenes Mediated Transformation of Isatis Tinctoria L. for the Efficient Production of Flavonoids and Evaluation of Antioxidant Activities

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC₅₀ values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.     

Metformin alleviates monoamine oxidase-related vascular oxidative stress and endothelial dysfunction in rats with diet-induced obesity

Molecular and Cellular Biochemistry - In the past decade, monoamine oxidase (MAO) with 2 isoforms, MAO-A and B, has emerged as an important source of mitochondrial reactive oxygen species (ROS) in...     

二角型小麦雄性不育系育性恢复性的研究

以5个同质异核二角型小麦雄性不育系ms(bicor-8222,ms(bicor)-83(37)65,ms(bicor)-偃师9号,ms(bicor)-80(6)及ms(bicor)-90-110为基本材料,与一批优良小麦品种（系）及部分亲本材料为父本进行测交,获得211个组合,考察其F1育性,结果表明;（1）5个同质异核不育系,除二角型非1B/1R不育系ms(bicor)-90-110与二角型1B/1R不育系ms(bicor)-83(37)65,ms(bicor)-偃师9号之间平均恢复度差异显著外,4个1B/1R不育系之间平均结实率差异不显著;（2）对同一不育系而言,与不同恢复系测交,其恢复度呈现连续变异;（3）同型非1B/1R不育系较1B/1R不育系恢复度普遍高;（4）对同一恢复系而言,各不育系测交F1的恢复度差异显著。     

数量性状主基因＋多基因混合遗传分析中鉴定多基因存在的IECM算法

为了进行２对主基因＋多基因混合遗传分析中的主基因存在的鉴定和多基因存在的鉴定以及多世代的联合遗传分析的分布参数估计,在ＥＣＭ算法和剖分成分分布方差为主基因变异组分、多基因变异组分和误差变异组分三部分基础上,提出了计算简便的迭代ＥＣＭ算法,简称ＩＥＣＭ算法,以利用 Ｐ_１、Ｆ_１、Ｐ_２和 Ｆ_（２：３）家系世代鉴定多基因存在为例阐明该算法．它的 ＣＭ步包含迭代ＣＭ_１、迭代ＣＭ_２和迭代ＣＭ_３步,在固定其它参数的情况下分别求分布平均数、多基因方差组分和误差方差的极大似然估计．通过１１３８－２ｘ邳县天鹅蛋杂交组合的Ｐ_１、Ｐ_２、Ｆ_１和Ｆ_（２：３）家系群体研究了大豆豆秆黑潜蝇的遗传规律．结果表明,它受 １对主基因的控制并有多基因的修饰．     

Continuous biosynthesis of abscisic acid (ABA) may be required for maintaining dormancy of isolated embryos and intact seeds of Euonymus alatus

Euonymus alatus (Thunb.) Sieb. is a popular landscape plant in the United States due to its brilliant red fall foliage. It is also an important ornamental plant in many other areas of the world such as China, Japan and Europe. However, E. alatus is considered as a highly invasive plant species in the US. Mutation breeding can be used to create sterile, non-invasive cultivars. Seeds are the most commonly used explants for mutagen treatments, but E. alatus mature seeds possess prolonged dormancy and only a low percentage of them germinate even after 18?months of cold stratification. Here we report an immature embryo culture method for E. alatus ??Compactus?? to circumvent the seed dormancy problem. Also, we have found that activated charcoal, gibberellic acid (GA₃) and 6-benzyladenine (BA) can reduce the dormancy of isolated embryos, which suggests that abscisic acid (ABA) might play a role in controlling seed dormancy. We have further demonstrated that exogenous ABA enhances dormancy of isolated E. alatus embryos while fluridone, an inhibitor for ABA biosynthesis, can effectively break their dormancy. These results, particularly the effect of fluridone, suggest that continuous ABA biosynthesis plays an important role in controlling the dormancy of E. alatus seeds.