Structure of the Small Outer Capsid Protein, Soc: A Clamp for Stabilizing Capsids of T4-like Phages

Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a “glue” between neighboring hexameric capsomers, forming a “cage” that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 Å resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.     

At the foot of the shrew: manus morphology distinguishes closely‐related Cryptotis goodwini and Cryptotis griseoventris (Mammalia: Soricidae) in Central America

Small‐eared shrews (Mammalia, Soricidae) of the New World genus Cryptotis are distributed from eastern North America to the northern Andes of South America. One well‐defined clade in this genus is the Central American Cryptotis mexicana group, whose members are set off from other species in the genus by their variably broader fore feet and more elongate and broadened fore claws. Two species in the C. mexicana group, Cryptotis goodwini Jackson and Cryptotis griseoventris Jackson, inhabit highlands in Guatemala and southern Mexico and are presumed to be sister species whose primary distinguishing feature is the larger body size of C. goodwini. To better characterize these species and confirm the identification of recently‐collected specimens, we obtained digital X‐ray images of the manus from large series of dried skins of both species. Measurements of the metacarpals and phalanges successfully separated most specimens of C. goodwini and C. griseoventris. These measurements also show that the fore feet of C. griseoventris from Chiapas, Mexico, are morphologically distinct from those of members of the species inhabiting Guatemala. Univariate, bivariate, and multivariate analyses indicate that fore foot characters are more conservative within species of the C. mexicana group than are cranio‐mandibular characters. Patterns of evolution of fore foot characters that superficially appear to be linear gradations are actually more complex, illustrating individual evolutionary trajectories. No claim to original US government works. Journal compilation © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 118–134.     

Genetic analysis of Ras signalling pathways in cell proliferation,migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras‐like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3‐kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D‐type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.     

Newly formed E‐cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes1

Background information. The appropriate regulation of cell–cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E‐cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin‐mediated cell–cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E‐cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell–cell contacts has been inferred from the presence of filopodia, the typical F‐actin structure induced by Cdc42 activation, as cells approach each other to establish cell–cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. Results. In the present study, we set out to address (a) whether Cdc42 is activated by new E‐cadherin cell–cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E‐cadherin clustering decreased Cdc42‐GTP levels. In the absence of serum‐ and migration‐induced Cdc42 activation, we demonstrated that cell–cell contacts do not induce filopodia or require Cdc42 function to assemble. Conclusion. We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re‐positioning or basolateral trafficking.     

退耕还林对宁南黄土丘陵区景观格局的影响——以中庄村典型小流域为例

"退耕还林"工程是关系到西部乃至中国生态恢复的重要工程。以黄土丘陵区"退耕还林"工程实施最早的典型小流域——中庄村小流域为研究区,应用景观生态学的原理与方法,选择斑块多样性、景观异质性和土地利用相对合理性等指数,对其退耕还林前(1993-2000年)后(2000-2005年)的变化情况进行分析,目的在于揭示"退耕还林"政策的实施对景观格局向良性演化的巨大推动作用。结果显示,退耕前(1993-2000年)研究区景观格局演变幅度很小,相应的,斑块多样性、景观异质性和土地利用相对合理性指数等景观指数的变化均较小。退耕后(2000-2005年),研究区发生了剧烈的景观格局演化,主要的景观变化过程是耕地转化为林地。此阶段研究区25°以上坡耕地基本退耕完毕,15-25°坡度范围是退耕还林的主要区域,其次为8-15°。相应的,2000-2005年小流域景观斑块总数减小,平均斑块面积增加,景观形状趋于复杂,斑块边界复杂性有所增加,景观斑块呈现团聚化的趋势。其中,耕地斑块总面积显著减小,斑块数下降,平均斑块面积略有增大;林地斑块总面积显著增加,斑块数保持稳定,景观优势度显著增加。土地利用相对合理性指数因此从1993年和2000年的0.668、0.664,显著上升至2005年的0.712。采用3期土地利用现状图对"退耕还林"实施前后两个阶段的景观格局演变过程进行分析,结果表明"退耕还林"是研究区景观格局良性演化的主要驱动力。     

羊布鲁氏菌非编码小RNA分子BSR-2的预测与鉴定

利用RT-PCR和RACE等方法对生物信息学预测的羊布鲁氏菌非编码小RNA(smallnon-codingRNA,sRNA)BSR-2进行了实验鉴定,并通过分析BSR-2在胞内生存缺陷株中的转录情况以及预测BSR-2的靶标基因对BSR-2的功能进行初步探讨.RT-PCR结果表明,BSR-2在羊布鲁氏菌的总RNA中存在转录本,而且在不同的应激条件下转录水平不同.RACE结果表明,BSR-2长224nt,位于Ⅱ号染色体的BMEII0742和BMEII0743之间的基因间区.进一步的实验结果表明,BSR-2可能与布鲁氏菌的胞内生存能力相关.     

小分子RNA抵御转座元件对基因组的侵袭

真核生物在漫长的进化繁衍过程中,一直处在抵御转座元件对基因组侵害的"斗争"中。在过去的十多年中,越来越多的证据指明,小分子RNA扮演了这样一个抵御转座子侵袭的角色。尽管这种抵御侵害的策略对于不同物种各具特点,但它们都呈现出惊人相似的共同特征。基本上,所有的机制都包含三个组成部分:首先,转座元件促使产生小分子RNA,在某些物种中主要是Piwi-interacting RNAs,而在其他物种中主要是small interfering RNAs;第二,作用于活跃转座子的小分子RNA通过RNA依赖性RNA聚合酶或切割机制进行扩增;第三,这些小分子RNA与含有Argonaute蛋白或Piwi蛋白的效应复合物相结合,从而作用于目标转座子的转录本,实现转录后沉默,或作用于目标转座子DNA,抑制染色质修饰和DNA甲基化。这些属性特征构成了一个限制由转座元件活动所造成的严重后果的系统,从而防止转座子侵袭所带来的突变积累,基因表达谱的改变,以及生殖腺发育不良和不育。     

动物snmRNA的系统识别与鉴定

小分子非编码RNA(snmRNA)在调控基因的转录和转录后加工、细胞分化和个体发育、遗传和表观遗传等几乎所有的重要生命活动中发挥关键作用。建立和发展snmRNA研究技术,系统地发现和注释基因组中的snmRNA基因并阐明其生物学意义是当前RNA组学的首要任务。围绕snmRNA的系统识别与鉴定等问题,该文对近年来采用实验技术和计算机预测方法发掘snmRNA所取得的主要研究成果进行综述。     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Morphology and molecular relationships of Leptofauchea rhodymenioides (Rhodymeniales,Rhodophyta), a new record for Japan

Leptofauchea rhodymenioides Taylor (Faucheaceae, Rhodymeniales) is reported from Japan for the first time, based on detailed morphological studies and molecular phylogenetic analyses of nuclear‐encoded small subunit ribosomal RNA (SSU rRNA) and plastid‐encoded rbcL gene sequences. This is the first report of male gametophytes and detailed carposporophyte development in the genus Leptofauchea. This species is characterized as follows: (i) flat, membranous, and regularly and dichotomously branched thalli; (ii) the older blades are constricted below the apices; (iii) the cortex is composed of a continuous layer with an irregularly arranged outer layer, and the medulla of two to three incomplete layers; (iv) gametophytes are dioecious; (v) in males, the cortical cells cut off two to three spermatangial mother cells, which produce terminal spermatangia; (vi) in females, the procarp is composed of a three‐celled carpogonial branch and a two‐celled auxiliary cell branch; (vii) upon fertilization, the carpogonium directly contacts the auxiliary cell; (viii) the auxiliary mother cell fuses with vegetative cells, and forms a large trunk‐like fusion cell; (ix) gonimoblast filaments develop outwardly, and transform completely into carposporangia; (x) the carposporophyte is covered with a pericarp with a well‐defined tela arachnoidea; (xi) the mature cystocarp is spherical, has an ostiole, and protrudes from the blade margins; and (xii) the cruciately divided tetrasporangia are formed in nemathecia, produced laterally from paraphyses or terminally on short filaments. Molecular analyses suggest that Leptofauchea forms a strong sister alliance with the genus Webervanbossea. The families Faucheaceae and Lomentariaceae, and the genera Leptofauchea and Webervanbossea are monophyletic, but the latter two genera are not included in the Faucheaceae.