Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyidimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m) = 8 +/- 0.4 degreesC). An increasing in T-m was also observed while the cationic lipid vesicles interacted with ssDNA.

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China

Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80-100 nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found. (c) 2007 Elsevier Inc. All rights reserved.

Direct visualization of the dynamics of membrane-anchor proteins in living cells

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane-anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane.

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmernbrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits. (c) 2006 Elsevier Inc. All rights reserved.

水稻小G蛋白Arf GTPase激活蛋白OsAGAP功能研究

　　锌指蛋白在植物生长发育中具有重要功能，它们可以识别并结合特定的DNA序列进行转录调控，还能够参与蛋白之间相互作用的调节。我们根据锌指蛋白等转录因子特征结构域的序列特点，从来自10 K水稻芯片的EST数据库中筛选出编码58个EST序列。通过对器官表达特异性的比较分析，从中选出七个只在单一器官表达的基因，并对这七个基因的功能进行研究。对其转基因水稻的表型分析发现，C1基因调节水稻的株高和穗的发育;LIM 家族的F9影响小花的形态，主要体现在雌蕊与雄蕊的发育;锌指蛋白S34调控叶倾角的变化;F14基因编码一个核定位的TFIIIA类锌指蛋白，具体功能尚不清楚;锌指蛋白F35转基因水稻主根缩短，侧根数目显著减少。它编码一个推测的ArfGAP (Arf GTPase activating protein)，据此我们将其命名为OsAGAP，并对其进行深入研究。 　　OsAGAP的cDNA全长为1328bp，编码的蛋白由320个氨基酸组成，含有两个保守结构域：锌指结构域和C2 结构域。其中锌指结构域属于CX2CX16CX2C类，即ArfGAP domain的特征结构。GTP酶活性测定试验表明，OsAGAP蛋白能够激活水稻Arf的GTP酶活性，另外，OsAGAP还能够恢复酵母ArfGAP缺失突变体的表型。说明OsAGAP编码的蛋白是水稻中的一个ArfGAP。 　　OsAGAP在水稻各器官中均有表达，但强弱有所不同。RNA原位杂交结果显示，它在茎尖分生组织与侧生原基及侧根部位表达强烈;它在根尖主要分布于中央维管组织、分生区、皮层细胞，最有趣的是恰好与生长素在根尖极性运输路径相吻合。在亚细胞水平，OsAGAP广泛分布于细胞膜、细胞质、细胞核。 　　OsAGAP超表达水稻主根、不定根长度缩短，侧根数目显著减少表现出类似于生长素极性运输突变体的表型。其主根伸长对TIBA的抑制作用不敏感，这暗示OsAGAP超表达水稻的生长素极性运输被破坏;另外，其对各种生长素的作用敏感性也发生变化，对IAA、2,4-D的不敏感，而对NAA的反应与野生型一致，根据各类生长素进出细胞机制不同，可以推测超表达水稻的输入能力存在缺陷。极性运输实验结果表明，超表达水稻极性运输能力被破坏;对生长素输入能力的测定进一步表明，超表达水稻根载体的介导的生长素输入能力显著下降。另外，NAA处理能够恢复超表达水稻中侧根发育受抑的表型缺陷。由此可见，OsAGAP在水稻中超表达破坏了生长素极性运输的输入能力。 　　FM1-43是一类特异标记囊泡运输的荧光染料。经其染色标记后，OsAGAP超表达水稻细胞内囊泡成片聚集，形成“BFA区间”，表现出囊泡运输被破坏的典型特征。透射电镜观察发现，超表达水稻细胞内有大量的小液泡，其中积累了电子密度很高的颗粒物质。由此推测，可能由于细胞的囊泡运输被破坏，导致胞内的代谢物质不能被正常运送或分泌，而在液泡中暂时贮存以维持细胞环境的稳定。 　　在酵母和动物细胞中的研究表明， ArfGAP是调控囊泡运输的一个重要因子，然而目前还没有关于ArfGAP在植物细胞中生理作用的报道。我们的结果说明，OsAGAP作为的一个ArfGAP，它通过调控水稻中的囊泡运输，而影响了生长素的极性运输，具体表现在对生长素输入能力的调控。由此，我们推测ArfGAP可能在生长素的极性运输中也起着重要的调控作用。 　　但OsAGAP在拟南芥中却通过调控植株生长素的水平，而影响了转基因拟南芥根的发育。每种生物都有多个ArfGAP，它们之间的分工存在联系，但各不相同。OsAGAP是拟南芥的外源基因，它在拟南芥中可能以不同于水稻的机制起作用。

水稻G蛋白偶联受体OSGPCR1功能的研究

水稻是世界上最重要的粮食作物，也是单子叶植物的模式植物，它为全球近一半的人口提供食物，但是低温、高盐、干旱等非生物胁迫，每年都会在全世界范围内造成水稻大面积减产。G蛋白介导的信号途径是传递胞外信号比较保守的作用机制之一。动物细胞对于G蛋白及其受体(GPCRs)的研究已经取得了很大的进展。而植物细胞中对它们的研究刚刚起步。本文从越冬稻低温响应芯片上筛选到一个膜蛋白，它编码一个推测的G蛋白偶联受体(G protein-coupledreceptor, GPCR)，据此我们将其命名为OsGPCR1，并对其进行深入研究。 OsGPCR1的cDNA全长为1407bp，编码468个氨基酸，在蛋白水平上的同源性比较结果显示，该基因与动物中研究的比较多的异源三聚体G蛋白偶联受体(G Protein- Coupled Receptor)同源性达到44%。经过跨膜结构域预测表明OsGPCR1具有9TMs结构，以GFP为标签的亚细胞定位表明OsGPCR1定位在膜上。GTP酶活性测定试验表明，OsGPCR1蛋白能够激活水稻RGA的GTP酶活性，此外，以泛素裂解体系为基础的酵母双杂交实验表明，OsGPCR1能够与RGA相互作用。说明OsGPCR1编码的蛋白是水稻中的一个G蛋白偶联受体。 OsGPCR1的表达受低温、干旱、高盐的诱导，但不受ABA，GA，ACC，IAA的诱导。在『F常生长条件下，OsGPCR1在水稻各器官中均有表达，但强弱有所不同。 在拟南芥和水稻中超表达OsGPCR1都能显著增强转基因植物对干旱、高盐、低温的耐受性。而在水稻中抑制OsGPCR1的表达，转基因水稻呈现出干旱、高盐、低温的敏感性。对转基因拟南芥下游基因的分析表明，超表达OsGPCR1能够在非胁迫条件下激活CBF途径中相关基因的表达。结合OsGPCR1不受ABA诱导的表达模式，我们推测OsGPCR1可能是通过不依赖于ABA这条途径而传递信号的。借助超表达和转反义水稻材料，利用水稻全基因组芯片研究OsGPCR1靶基因的结果表明，不论OsGPCR1基因表达量的降低或上升，都导致大约30%的与转运相关的基因的表达量发生改变。这暗示OsGPCR1可能通过囊泡运输传递胞外信号。此外FM4-64对超表达和转反义水稻幼根细胞染色标记后，OsGPCR1反义抑制水稻细胞内囊泡在细胞两端呈聚集状，即形成“BFA区间”，而超表达OsGPCRI水稻细胞内囊泡呈密集状。这些结果都表明OsGPCR1可能通过调控囊泡运输而将胞外胁迫信号传递进胞内。

Proteomic Analysis of Proteins Involved in Spermiogenesis in Mouse

Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

New symbiotic hypothesis on the origin of eukaryotic flagella

The origin of eukaryotic flagella has long been a mystery. Here we review the possibility that flagella sprouted evolutionarily from the eukaryotic cell proper seems very unlikely because it is hard to imagine what function and benefit in natural selection the flagella would have provided to the cells when they first emerged as simple buds. Lynn Margulis' 1970 spirochete hypothesis, though popular still, has never been confirmed. Moreover, the absence of tubulin and axonemal dynein in the spirochetes and the incapability of the bacterial and eukaryotic membranes' making a continuum now suggest that the hypothesis is outdated. Tubulin genes were recently identified in a new bacteria division, verrucomicrobia, and microtubules have also been found in one of these species, epixenosomes, the defensive ectosymbionts. On the basis of these data, we propose a new symbiotic hypothesis: that the mid-ancestor of eukaryotic cells obtained epixenosomelike verrucomicrobia as defensive ectosymbionts and the ectosymbionts later became endosymbiotic. They still, however, protruded from the surface of their host to play their role. Later, many genes were lost or incorporated into the host genome. Finally, the genome, the bacterial membrane, and the endosymbiotic vesicle membrane were totally lost, and fingerlike protrusions with microtubules formed. As the cells grew larger, the defensive function of the protrusions eventually weakened and then vanished. Some of the protrusions took on a new role in cell movement, which led them to evolve into flagella. The key step in this process was that the dynein obtained from the host evolved into axonemal dyneins, attaching onto the microtubules and forming motile axonemes. Our hypothesis is unproven, but it offers a possible explanation that is consistent with current scientific thought. We hope that our ideas will stimulate additional studies on the origin of eukaryotic flagella and on investigations of verrucomicrobia. Whether such studies confirm, refine, or replace our hypothesis, they should nevertheless further our understanding of the origin of eukaryotic cells.

Distribution of rDNA in the nucleus of Giardia lamblia - Detection by Ag-I silver stain

OBJECTIVE: To determine whether rDNA of Giardia lamblia forms a nucleolus organizer region (NOR)-like structure and is in a very primitive state. STUDY DESIGN: G lamblia was used as the experimental animal, with Euglena gracilis as the control. The distribution was demonstrated indirectly by the modified Ag-I silver technique, which can specifically indicate the NOR under both light and electron microscopes. RESULTS: In the ultrathin sections of silver-stained Euglena cells, all the silver grains were concentrated in the fibrosa of the nucleolus, while no grains found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver-stained Giardia cells, no nucleolus was found; a few silver grains were scattered in the nucleus but were not concentrated in any specific region. CONCLUSION: The distribution of silver grains in G lamblia showed that the transcription of rDNA occurs inside the nucleus, though no nucleolus is present. It is possible that chromosomes are in a very primitive state in diplomonad cells; as each chromosome has few prRNA genes, the transcription is independent of a nucleolus. These results imply that the rDNA of Giardia does not form a NOR-like structure and seems to represent a very primitive state in the evolution of the nucleolus.

Differential expression of the Brunol/CELF family genes during Xenopus laevis early development

The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes starts at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed in both the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 are only present in the cytoplasm, indicating their different functions.