Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (K-d) of 880 nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400 nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott-Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

小麦TaRanBP基因功能的初步分析

小G蛋白（small GTPases）是真核生物中广泛存在的一类调节各种生命活动的信号分子。根据结构与功能的不同，小G蛋白家族成员可分成五个亚家族，分别为Ras，Rab，Rho，Arf和Ran。五类小G蛋白通过其活化态（GTP结合态）和非活化态（GDP结合态）的相互转换行使着各种功能。Ras GTPases在酵母和哺乳动物中调节细胞增殖过程; Rho GTPases调控肌动蛋白重组过程，并参与MAP 激酶的细胞信号转导过程等; Rab GTPases和Arf GTPases分别在膜转运过程中起着不同的重要作用;而Ran GTPases则在核孔位置调节着蛋白和RNA分子的运输过程。 小G蛋白附属蛋白调节着小G蛋白活化态与非活化态之间的转换，其中鸟核苷酸交换因子(guanine nucleotide exchange factors, GEFs)可以催化小G蛋白转换为GTP结合形式，即活化态;而GTPase 激活蛋白(GTPase-activating proteins, GAPs)和小G蛋白结合蛋白(small GTPases binding proteins)可以激活小G蛋白自身的水解活性，从而将其转变成非活化态形式。 相比其它小G蛋白，Ran GTPases及其附属蛋白在真核生物中的研究相对较少。已有的成果表明它们主要在核质运输过程中及对相应的信号转导途径起调节作用。而针对Ran GTPases及其附属蛋白在真核生物尤其是高等植物个体发育过程中的作用，目前报道还很少。 为了揭示Ran结合蛋白(Ran binding protein, RanBP)在植物发育过程中的作用，本文通过转基因手段对其功能进行 了研究。在此之前，本实验室已从小麦cDNA文库中成功克隆Ran结合蛋白基因：TaRanBP。该基因cDNA全长1035 bp，编码207个氨基酸。通过农杆菌介导叶圆片法，分别用正义、反义及TaRanBP与GFP融合蛋白等表达载体转化烟草，并成功获得转基因植株。亚细胞定位观察发现TaRanBP蛋白主要定位于细胞质内，尤其是在核膜附近富集。生理学和细胞学等方面的研究分析发现，TaRanBP基因在烟草个体发育过程中产生重要作用。过量表达TaRanBP基因的转基因植株在一定数量上表现出愈合的花冠筒上出现不同程度开裂，花冠筒上有附生舌状花瓣，及带有花瓣状颜 色的花萼等异常花表型。同时，转反义基因在一定程度上促进了转基因植株初生主根的生长（为对照烟草的2.3倍），而转正义基因烟草与对照烟草的初生主根长度差异不明显。用碘化丙锭(Propidium Iodide, PI)进行根部细胞染色。观察发现，不同的转基因烟草与对照烟草之间在根的各个不同形态区域的细胞大小差异不明显，推测根长的差异可能是由于整体细胞数目变化的原因导致。向重力性实验发现，转反义基因烟草幼苗较对照烟草的向重力性反应增加，而转正义基因的则表现为降低。激素吲哚乙酸(Indoleacetic Acid, IAA)的添加处理可以恢复转反义基因烟草的向重力性异常表型，而对转正义基因烟草几乎无影响。添加激动素(Kinetin, KT)的处理发现不同转基因烟草和对照烟草的向重力性均有减弱。观测后期，转正义基因的向重力敏感性较对照烟草得到恢复。测量不同转基因株系T1代幼苗鲜重，发现不同转基因烟草和对照烟草的幼苗鲜重动态变化在各个时间点有差异，且差异情况不尽相同。而不同转基因幼苗T1代幼苗可溶性蛋白含量较对照烟草有不同程度的下降。这种下降并没有影响转基因烟草的整体生长进程，开花期和结实情况与对照烟草相比也无明显变化。

小麦细胞壁钙调素和钙调素结合蛋白的研究

本实验以小麦黄化胚芽鞘为材料首次从生化上证明细胞壁CaM的存在，并发现在小麦细胞壁中存在除CaM以外的另一种钙结合蛋白和CaM结合蛋白。主要实验结果如下： 1、根据小麦细胞壁提取液中存在具热稳定性;能与动物CaM抗体发生免疫交叉反应;依赖Ca++激活牛脑PDE及其激活受CaM抑制剂CPZ抑制等特性的物质，鉴定出在小麦细胞壁中存在CaM。 2、小麦细胞壁CaM以水溶性和离子键结合于细胞壁的两种形式存在，但主要以离子键结合于细胞壁的形式存在。每克鲜重小麦黄化胚芽鞘中，离子键结合于细胞壁的CaM占细胞壁总CaM的86.4%，细胞壁总CaM占胞内CaM的2.7%。 3、根据小麦细胞壁CaM依赖Ca++与苯基疏水结合;在紫外外吸收光谱上具有五个特征吸收峰;在有钙和缺钙情况下SDS电泳图谱上呈现不同的迁移率;对牛脑PDE的激活剂量反应和激活PDE时对Ca++的敏感性等特性，证明与胞内CaM具有相同的理化特性。 4、小麦细胞壁中存在9种CaM结合蛋白（或亚基），其中以分子量为40700的CaM结合蛋白（或亚基）含量最高。这些CaM结合蛋白中不存在过氧化物酶、ATP酶和酸性磷酸酯酶的活性。 5、小麦细胞壁中存在除CaM以外的另一种钙结合蛋白，分子量为38000。

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.

Identification of genes differentially expressed in wild type and Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin -28k, paravalbumin, matrix gamma-carboxygluta mate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.

Cloning and expression of medaka dazl during ernbryogenesis and gametogenesis

The Deleted in azoospermia family consists of RNA-binding proteins Bottle, Daz, and Daz-like (Dazl) that are expressed in the germline. Here, we report the cloning and expression of the medakafish (Oryzias latipes) dazl gene (odazl). Interestingly, although the predicted medaka Dazl protein (oDazl) contains a RRM motif and a DAZ repeat characteristic of its mammalian homologs, it lacks 80 aa at the C-terminus. By RT-PCR, RNA in situ hybridization, Western blotting and fluorescent immunohistochemistry using a rabbit anti-DazI antibody (alpha Oazl), we analyzed the expression patterns of odazl and its protein. The odazl transcript persists throughout embryogenesis and delineates with primordial germ cells. In adults, the expression of odazl RNA and its protein is restricted to germ cells of both the testis and ovary. We observed differential expression of RNA and protein at critical stages of gametogenesis. In the testis, the odazl RNA is low at premeiotic stages, abundant at meiotic stages, but absent in postmeiotic stages; whereas the oDazl protein is rich in premeiotic stages, reduced at meiotic stages, becomes barely detectable or absent in postmeiotic round spermatids or sperm, respectively. This is in sharp mature spermatozoa. In the ovary, the odazl RNA contrast to the human situation where the Dazl transcript and protein are present in and protein persist throughout oogenesis and also show differential expression at premeiotic, meiotic and postmeiotic stages. Thus, the odazl or its protein is a marker for germ cells during embryogenesis and at critical stages of gametogenesis in both sexes of medaka. (c) 2006 Elsevier B.V. All rights reserved.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Isolation of the main light-harvesting chlorophyll a/b-protein complex from thylakoid membranes of marine alga, Bryopsis corticulans by a direct method

The main chlorophyll a/b light-harvesting complex (LHC 11) has been isolated directly from thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion exchange and gel-filtration chromatography. LHC 11 proteins in the membrane extracts treated with 3% n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and oligomeric subcomplexes of LHC 11 have been obtained by fractionation of the LHC 11 mixes with sucrose density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption spectrum showed that LHC 11 monomers, trimers and oligomers prepared through this work were intact and in high purity. Our report is the first to show that it is possible to purify LHC If directly from thylakoid membranes without extensively biochemical purification.

Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play Important roles in innate immunity In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5'-untranslated region (UTR) of 101 bp and a 3'-UTR of 164 bp The deduced amino acid sequence of SmLec1 shares 34%-38% overall identities with the C-type lectins of several fish species In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus uncle The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 degrees C) Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection (C) 2010 Elsevier Ltd. All rights reserved