A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity - The first enzyme in the viral mRNA capping pathway

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640 1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins. (C) 2015 Elsevier Inc. All rights reserved.

Uso de células iPS en terapia regenerativa renal: obtención de células iPS mediante vector no integrativo y diferenciación hacia podocitos.

[ES]En las sociedades modernas existe una creciente preocupación por el aumento de la incidencia de la enfermedad renal crónica. Debido a la deficiencia de donantes de órganos y al elevado coste del tratamiento de diálisis, existe la necesidad de desarrollar nuevos tratamientos para estos pacientes. La medicina regenerativa basada en la aplicación de células iPS es una opción prometedora para el tratamiento de esta enfermedad. Sin embargo, la falta de conocimientos sobre el estado pluripotencial de las células y sobre su proceso de diferenciación, así como las limitaciones derivadas del propio procedimiento de reprogramación, impiden su aplicación clínica en un futuro inmediato. Para que se convierta en realidad, numerosas investigaciones se están llevando a cabo con el objetivo de mejorar el procedimiento y hacerlo adecuado para su aplicación clínica. En este trabajo se propone un método que permitiría obtener células iPS a partir de células mesangiales mediante la transfección con un vector no integrativo, el virus Sendai, portador de los genes Oct3/4, Sox2, Klf4 y c-Myc. Al tratarse de un vector no integrativo, se minimizaría el efecto del proceso de reprogramación sobre la estabilidad del genoma celular. Además, en este proyecto se estudiará la capacidad de las células iPS obtenidas para diferenciarse en células progenitoras de podocitos que puedan ser aplicadas específicamente en terapias regenerativas para enfermos renales crónicos.

Valoracion de alternativas en paneles vegetales prefabricados

En las sociedades modernas existe una creciente preocupación por el aumento de la incidencia de la enfermedad renal crónica. Debido a la deficiencia de donantes de órganos y al elevado coste del tratamiento de diálisis, existe la necesidad de desarrollar nuevos tratamientos para estos pacientes. La medicina regenerativa basada en la aplicación de células iPS es una opción prometedora para el tratamiento de esta enfermedad. Sin embargo, la falta de conocimientos sobre el estado pluripotencial de las células y sobre su proceso de diferenciación, así como las limitaciones derivadas del propio procedimiento de reprogramación, impiden su aplicación clínica en un futuro inmediato. Para que se convierta en realidad, numerosas investigaciones se están llevando a cabo con el objetivo de mejorar el procedimiento y hacerlo adecuado para su aplicación clínica. En este trabajo se propone un método que permitiría obtener células iPS a partir de células mesangiales mediante la transfección con un vector no integrativo, el virus Sendai, portador de los genes Oct3/4, Sox2, Klf4 y c-Myc. Al tratarse de un vector no integrativo, se minimizaría el efecto del proceso de reprogramación sobre la estabilidad del genoma celular. Además, en este proyecto se estudiará la capacidad de las células iPS obtenidas para diferenciarse en células progenitoras de podocitos que puedan ser aplicadas específicamente en terapias regenerativas para enfermos renales crónicos.

Fast and Efficient Neural Conversion of Human Hematopoietic Cells

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

Administração de cafeína durante a gestação altera parâmetros testiculares na prole de camundongos C57/BL6 na vida adulta

Metabólitos ativos da cafeína apresentam toxicidade, podendo causar efeitos deletérios em muitos órgãos no período fetal pelo consumo materno. O estudo objetivou avaliar o papel da administração de cafeína durante a gestação em vários parâmetros importantes para a função testicular em camundongos adultos C57BL/6. Fêmeas grávidas foram divididas em: grupo controle (C), que receberam injeções de sol. salina (0,9% NaCl) e grupo cafeína (CF), que receberam diariamente injeções subcutâneas de 20 mg / kg de cafeína. Filhotes tiveram livre acesso à ração padrão desde o desmame até três meses de idade, quando foram mortos. O grupo CF apresentou uma redução no consumo alimentar (C = 4,36 0,14; CF = 3,79 0,05/g, p<0,05) e ganho de massa corporal (C = 25,73 0,14; CF = 21,08 0,41/g, p=0,0001). Ainda este grupo, apresentou alterações estruturais nos testículos da prole, como redução no peso relativo (C = 0,078 0,003; CF = 0,063 0,002/g, p=0,002), diâmetro tubular (C = 455,4 2,7; CF = 393,5 3,1/m, p=0,0001), lume tubular (C = 310,6 2,5; CF = 254,8 2,9/m, p=0.0001) e altura do epitélio seminífero (C = 144,8 0,9; CF = 138,7 1,3/m, p=0,0005) observados pela técnica de morfometria. Testosterona sérica avaliada por quimioluminescência foi reduzida (C = 6,6 2,8; CF = 0,5 0,2/ng/ml, p=0.04). Western Blotting foi utilizado para análise de expressão protéica. Houve aumento do receptor de leptina (C = 0,81 0,04; CF = 1,28 0,15/g, p=0,01), do receptor para o hormônio leteinizante (C = 0,92 0,05; CF = 0,74 0,05/g, p=0,01),da aromatase (C = 0,32 0,03; CF = 0,48 0,05/g, p=0,03) e do regulador pró apoptose (C = 0,27 0,02; CF = 0,42 0,03/g, p=0,01) enquanto o receptor para hormônio folículo estimulante (FSHR) (C = 0,76 0,06; CF = 0,56 0,04/g, p=0,02), o receptor para proteína reguladora da esteroidogênese (C = 1,009 0,065; CF = 0,584 0,060/g, p=0,0007), a proteína do fator de crescimento vascular endotelial (C = 0,33 0,08; CF = 0,05 0,01/g, p=0,009), o receptor de androgênio (C = 0,97 0,11; CF = 0,59 0,07/g, p=0,02) e o antígeno nuclear de proliferação celular (C = 0,93 0,11; CF = 0,48 0,07/g, p=0,01) foram reduzidos. Este estudo sugere que o consumo de cafeína durante a gravidez parece ser nocivo à fertilidade de animais machos, caracterizando o fenômeno de programação, o que gera alterações funcionais e estruturais nos testículos da prole adulta.

人、黑猩猩和叶猴FKN全基因的克隆及体外表达的比较研究

下载PDF阅读器目的:克隆人、黑猩猩和叶猴FKN全基因及体外表达,比较研究FKN在进化过程中基因组水平和蛋白表达水平的差异.方法:应用基因重叠延伸拼接PCR法(Gene splicing by overlapping extension PCR,SOE-PCR)将FKN的3个外显子编码序列依次进行前后拼接,然后插入pcDNA3.1/myc-His(-)A真核表达载体中,经酶切、测序鉴定后转染CHO细胞体外表达,RT-PCR、SDS-PAGE和Western blot检测其表达产物.结果:酶切、测序鉴定证实插入的基因片段为完整的FKN,RT-PCR可从转染的CHO细胞中扩增出一条与目的基因大小一致的DNA片段,其表达蛋白能分泌至胞外,SDS-PAGE显示其分子量约为95 000,抗c-myc抗体可与载体上的c-myc蛋白特异性结合.测序显示人、黑猩猩和叶猴相比,FKN基因除了有散在的点突变外,还发现有一明显的30 bp的缺失,但此缺失对FKN蛋白的表达并不影响.结论:成功克隆人、黑猩猩和叶猴FKN全基因,基因组水平和蛋白表达水平的比较研究为后续探讨FKN在高级灵长类物种进化过程中免疫学功能的演变奠定基础.

核基因在鸟类分子系统发育学研究中的应用

核基因作为一种新的遗传标记,近年来被广泛应用于鸟类分子系统发育研究中.核基因与线粒体基因位于不同的遗传载体上,因此被引入到系统发育学研究中为物种树的重建提供独立的证据.常用的外显子标记为重组激活基因1(RAG-1),重组激活基因2(RAG-2),癌基因c-myc,原癌基因c-mos,它们由于缓慢的进化速率而被用于鸟类高级分类阶元的系统学研究中.常用的内含子标记是β纤维蛋白原基因内含子7(β-fibrinogen intron7,β-fibint7),肌红蛋白基因内含子Ⅱ(myoglobin intionⅡ).内含子标记通常与线粒体序列联合使用,形成具有互补系统发育信号的数据集,应用于各种分类阶元的系统学研究中.

中国鸟类的DNA分类及系统发育研究概述

鸟类分类是鸟类学其他研究领域的基础,近年来分子技术的发展,以及计算机技术的应用为鸟类分类学和鸟类系统演化研究提供了新的研究手段,给传统的系统分类研究带来了新的机遇.Tautz等于2002年首先提出运用DNA序列作为生物分类系统的主要平台,即DNA分类学(DNA Taxonomy).而Hebert等于2003年则首次提出了DNA条形码(DNA Barcoding)的概念,并对其物种分类和鉴定意义予以肯定,建议利用线粒体细胞色素C氧化酶亚单位Ⅰ(COI)的特定区段来做DNA条形编码的基础.在鸟类DNA分类方面,国内学者应用线粒体基因Cut b,COI,c-mos,c-myc,12s rRNA,16s rRNA,ND2,ND3,CR,RAG-1以及核基因myoglobin introⅡ等不同片段对很多类群进行了分类探讨和系统发育研究.但是主要集中在鸡形目及雀形目鸟类.中国是鸟类多样性极其丰富的国家,近年来很多亚种、种及以上分类阶元依然存在问题,因此,中国鸟类物种的分类地位、系统发育与演化关系等依然有很多问题等待深入研究.目前国内基于COI的鸟类分类及系统发育研究有了一些报道,但是真正的DNA条形码工作尚需继续、深入地开展.

Analysis of steric mass-action model for protein adsorption equilibrium onto porous anion-exchange adsorbent

The ion-exchange equilibrium of bovine serum albumin (BSA) to an anion exchanger, DEAE Spherodex M, has been studied by batch adsorption experiments at pH values ranging from 5.26 to 7.6 and ionic strengths from 10 to 117.1 mmol/l. Using the unadjustable adsorption equilibrium parameters obtained from batch experiments, the applicability of the steric mass-action (SMA) model was analyzed for describing protein ion-exchange equilibrium in different buffer systems. The parametric sensitivity analysis was performed by perturbing each of the model parameters, while holding the rest constant. The simulation results showed that, at high salt concentrations or low pHs close to the isoelectric point of the protein, the precision of the model prediction decreased. Parametric sensitivity analysis showed that the characteristic charge and protein steric factor had the largest effects on ion-exchange equilibrium, while the effect of equilibrium constant was about 70%-95% smaller than those of characteristic charge and steric factor under all conditions investigated. The SMA model with the relationship between the adjusted characteristic charge and the salt concentration can well predict the protein adsorption isotherms in a wide pH range from 5.84 to 7.6. It is considered that the SMA model could be further improved by taking into account the effect of salt concentration on the intermolecular interactions of proteins. (c) 2006 Elsevier Ltd. All rights reserved.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

人类基因组中保守二级结构的纯净化选择及其在转录调控网络中的作用

保守序列是一种跨物种保守的基因组序列，而且绝大多数为非蛋白编码序 列。保守序列在人类遗传疾病中发挥着重要作用。其中，一部分保守序列能够 折叠形成二级结构。已鉴定的一些保守二级结构编码一些RNA 分子，如 microRNA、RNA 编辑序列和组蛋白mRNA 3’端非翻译区茎环结构等。但是，对 于绝大部分的保守二级结构，它们的生物学功能以及作用于它们上面的进化作 用力依然是未知的。 群体的SNP 数据在分析序列上的进化作用力时非常有效。SNP 在群体中的 频率会因为受到不同的进化作用力而表现出差异，而与其是否位于基因组中的 突变热点无关。对于受纯净化选择作用的SNP，它们的频率一般会比中性SNP 具有低的新生型等位基因频率（DAF）。我们运用生物信息学的方法，在人类基 因组保守二级结构中找到746 个SNP。这746 个SNP 与基因组其它区段的SNP 在突变模式上并不存在显著差异，在保守二级结构内同样存在突变热点。通过 与侧翼序列SNP 的分布比较发现，保守二级结构上SNP 密度约为其侧翼序列的 2/3。相比于侧翼序列SNP，有更高比例的保守二级结构SNP 具有低的DAF 值。 这些结果提示，有很多保守二级结构上的SNP 因为受到纯净化选择作用而在现 代人群中被剔除了。保守二级结构与侧翼序列在SNP 密度和DAF 上的差异要高 于保守序列与非保守序列之间的差异，提示保守二级结构是受到纯净化选择作 用最为严格的一类保守序列。我们发现，在保守二级结构内部，纯净化选择作 用的强度也有差异。茎区比环区具有更低的SNP 密度，而且有更高比例的茎区 SNP 具有低的DAF 值。这个结果提示，保守二级结构上的纯净化选择力主要作 用于茎区上的位点。我们推测，这可能是茎区上的突变往往比环区的突变对二级结构的造成更大的影响导致的。 我们通过寻找保守二级结构与转录因子SOX2、OCT4、NANOG、SUZ12 和C-MYC 结合位点之间的重叠，还分析了保守二级结构在转录调控网络中的作用。结果 显示，很多保守二级结构是作为转录因子的结合位点调控了许多与发育相关的 转录因子编码基因的表达。转录因子与保守二级结构之间的结合模式非常复杂， 可以有多个转录因子结合到同一个保守二级结构上，也可以是一个转录因子结 合到自身编码基因相关的保守二级结构上。不同的转录因子和保守二级结构结 合可以主导靶基因的特异模式，当绝大多数相关的保守二级结构与SUZ12 结合 时，基因表达受到抑制，而当绝大多数相关的保守二级结构不与SUZ12 结合时， 基因表达受到激活。在转录调控网络中，约有30%的保守二级结构是作为启动 子来调控基因的表达。因为转录因子SOX2、OCT4、NANOG、SUZ12 和C-MYC 仅仅 只结合到很小一部分保守二级结构上，提示可能还有更多的转录因子会结合到 保守二级结构上。因此，保守二级结构介导的转录调控网络要比目前已知的复 杂得多。

Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri

Thioester-containing proteins are a family of proteins characterized by the unique intrachain beta-cysteinyl-gamma-glutamyl thioester, which play important roles in innate immune responses. The cDNA of Zhikong scallop Chlamys farreri thioester-containing protein (designated as CfTEP) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTEP was of 4616 bp, consisting of a 5 '-terminal untranslated region (UTR) of 30 bp and a 3 ' UTR of 140 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The CfTEP cDNA encoded a polypeptide of 1481 amino acids with the theoretical isoelectric point of 5.98 and the predicted molecular weight of 161.4 kDa. The deduced amino acid sequence of CfTEP contained the canonical thioester motif GCGEQ, nine potential N-glycosylation sites and a C-terminal distinctive cysteine signature. It also contained a presumed catalytic histidine and proteolytic cleavage sites that were similar to C3 molecules. The high similarity of CfTEP with the thioester-containing proteins in other organisms, such as the TEPs from insects, the complement component C3, C4, C5 and the protease inhibitor alpha(2)-macroglobulin indicated that CfTEP should be a member of TEP family. The phylogenetic analysis revealed that CfTEP was closely related to TEPs from mollusc, nematodes and insects, and they formed a separate branch apart from the branches of complements factors and alpha(2)-macroglobulins. The spatial expression of CfTEP transcripts in healthy and bacterial challenged scallops was examined by semi-quantitative RT-PCR. The CfTEP transcripts were mainly detected in the tissues of hepatopancreas and gonad, and remarkably up-regulated by Microbial challenge, which suggested that CfTEP was a constitutive and inducible acute-phase protein involved in immune defense. These results provided new insights into the role of CfTEP in scallop immune responses, as well as the evolutionary origin of this important, widespread and functionally diversified family of proteins. (c) 2007 Published by Elsevier Ltd.

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

New wild Brassica oleracea L. germplasm at its western distribution limit in the Iberian Peninsula

An exploration and collection mission for wild Brassica oleracea populations was carried out in spring and summer of 2013. The aim of this collection was to expand the number of accessions of wild Brassica oleracea available for basic and applied research in plant breeding. In this paper we report a new accession of wild Brassica oleracea in an unexplored coastal area of Galicia, NW Iberian Peninsula. Details of population ecology and vegetation, soil, climate and geographic data were recorded for this population. The “Endangered” threat category for the region is proposed, and actions for in situ and ex situ conservation are proposed. Seeds will be added to the germplasm collections of University of Santiago de Compostela and Misión Biológica de Galicia (CSIC) for further research on diverse aspects of the dynamics and ecophysiology of the population along with characterization and evaluation of useful traits.