Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

New sphingolipids with a previously unreported 9-Methyl-C-20-sphingosine moiety from a marine algous endophytic fungus Aspergillus niger EN-13

Asperamides A (1) and B (2), a sphingolipid and their corresponding glycosphingolipid possessing a hitherto unreported 9-methyl-C-20-sphingosine moiety, were characterized from the culture extract of Aspergillus niger EN-13, an endophytic fungus isolated from marine brown alga Colpomenia sinuosa. The structures were elucidated by spectroscopic and chemical methods as (2S,2'R,3R,3'E,4E,8E)-N-(2'-hydroxy-3'-hexadecenoyl)-9-methyl-4,8-icosadien-1,3-diol (1) and 1-O-beta-D-glucopyranosyl-(2S,2'R,3R,3'E,4E,8E)-N-(2'-hydroxy-3'-hexadecenoyl)-9-methyl-4,8-icosadien-1,3-diol (2). In the antifungal assay, asperamide A (1) displayed moderate activity against Candida albicans.

栉孔扇贝(Chlamys farreri)Toll样受体及其信号传递相关基因的克隆与表达

扇贝是我国海水养殖的重要品种，但自1994年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。引起扇贝大规模死亡原因是多方面的，其主要原因是养殖环境恶化、扇贝种质衰退和抗病力下降。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。 Toll样受体（TLRs）家族是新近发现的模式识别受体（PRRs），参与识别病原体相关的分子模式（PAMPs），在天然免疫系统中起着非常重要的作用。哺乳动物中Toll样受体信号通路还参与诱导树枝状细胞成熟、参与免疫耐受、参与凋亡发生发展、介导非感染性因素的识别等，被视为联系天然免疫和获得性免疫的桥梁。同时果蝇的Toll信号通路也是不具备获得性免疫的果蝇赖以抵御病毒、细菌和真菌感染，介导天然免疫反应的重要信号通路。 本研究采用大规模EST测序方法，结合Genome Walker库的构建和cDNA末端快速扩增技术，从栉孔扇贝克隆得到CfToll-1、CfMyd88、CfTRAF6和CfCactus这四个Toll样受体信号通路基因的全长cDNA，同时用荧光实时定量PCR技术检测了这些基因的组织分布及在脂多糖（LPS）和肽聚糖（PGN）刺激下的表达规律。 栉孔扇贝Toll样受体（CfToll-1）的cDNA序列全长4308 bp，包含5’非翻译区（UTR）211 bp，3597 bp的开放阅读框，500 bp的3’UTR，最后为18个腺嘌呤的ploy A 尾巴。开放阅读框编码1198个氨基酸的多肽，该多肽的估计分子量为137.41kd，估计的等电点为5.62，该多肽有信号肽，具有一个预测的跨膜区，因此是一种跨膜蛋白。经BLAST比对，CfToll-1基因与节肢动物多种Toll蛋白高度的相似性。SMART（Simple Modular Architecture Research Tool）软件分析，CfToll-1包含典型的Toll样受体的结构：富含亮氨酸的重复序列的胞外区（leucine-rich repeats, LRR），一段跨膜结构域，以及胞内区的TIR结构域（Toll/IL-1 receptor homologous region）。利用Real-time RT-PCR发现CfToll-1mRNA在扇贝体内普遍存在于血细胞、肌肉、外套膜、心、性腺和鳃组织中。利用体外培养的原代血细胞系研究不同浓度LPS刺激后CfToll-1的表达变化，结果显示低剂量（100ng.mL-1 ）LPS 使CfToll-1 mRNA表达量减小，该变化在1.5h、3h 和9h组差异显著，虽然在6h组表达量稍有恢复，但尚未达到对照水平；用1μg.mL-1LPS处理细胞时， 6h组CfToll-1表达量明显上调，约为对照水平的2倍。证实细菌结构脂多糖对CfToll-1基因的表达有影响，且这种影响有剂量依赖效应。 栉孔扇贝Myd88同源基因（CfMyd88）的cDNA序列全长1554bp，包含5’UTR 427 bp，1101bp的开放阅读框，最后为18个腺嘌呤的ploy A 尾。CfMyd88的开放阅读框可编码367个氨基酸的多肽，该多肽的估计分子量为42.37kD，估计的等电点为5.71。利用SMART程序分析发现CfMyd88编码了Death和TIR结构域, 这两个结构域是Myd88特征结构。BLAST程序发现扇贝的序列与数据库哺乳动物的Myd88基因高度同源。原代培养的扇贝血细胞在受到PGN刺激后，CfMyd88 mRNA表达在1.5小时开始下调，直到9小时下调至对照表达量的1/10，证实肽聚糖结构对CfMyd88基因的表达有影响。 栉孔扇贝TRAF6同源基因（CfTRAF6）的cDNA序列全长2510bp，包含5’UTR 337 bp，1965bp的开放阅读框，3’UTR 208bp，最后为21 个腺嘌呤的ploy A 尾巴。CfTRAF6开放阅读框编码655个氨基酸的多肽，该多肽的估计分子量为74.09kD，估计的等电点为6.01。InterPro Scan在线分析发现CfTRAF6有典型的TRAF蛋白家族的特征结构，包括的一个指环结构，两个锌指结构，一个MATH （the meprin and TRAF homology）结构域以及Coiled-coil区域。CfTRAF6的序列与数据库多物种的TRAF6高度同源，同源性最高的是乌贼序列（Identity＝68）和鼠类（Identity＝45%）。利用Real-time RT-PCR，发现CfTRAF6在各组织普遍存在，在性腺中的表达最高。原代培养的扇贝血细胞在受到不同浓度PGN刺激后，与CfMyd88的情况一样，CfTRAF6的表达量变化减少，且这种变化随剂量的增加更加明显。 栉孔扇贝Cactus同源基因（CfCactus）的cDNA序列全长2488bp，包含5’UTR 181 bp，840bp的开放阅读框， 3’UTR 1467bp，最后为19个腺嘌呤的ploy A 尾巴。CfCactus的开放阅读框编码279个氨基酸的多肽，该多肽的估计分子量为31.37 kD；估计的等电点为4.74，与果蝇的Cactus基因的等电点相近（4.5）。利用SMART程序分析发现CfCactus主要编码了ANK结构域（ankyrin repeats）。Cactus基因为哺乳动物NF-κB抑制蛋白IκB的同源分子，BLAST 程序发现扇贝的序列与数据库多物种的Cactus或IκB基因高度同源。同源性最高的是太平洋牡蛎（Identity=35%）和圆尾鲎（Identities = 44%）。对CfTCactus mRNA在扇贝的血细胞、性腺、 肠的组织表达进行分析，并同时与CfTRAF6和CfMyd88的表达量进行了对比，发现CfCactus的表达水平明显高于这两个基因，而且CfTRAF6的基因表达量也高于CfMyd88，表现出级联放大效应。正常情况下，三个基因在性腺的表达量最高，推测这条通路可能和发育等功能密切相关。 通过本研究我们首次在双壳类软体动物找得到与果蝇Toll蛋白家族高度同源的CfToll-1基因，同时发现其他三个在Toll样受体信号传递过程中起重要作用的基因，其中包括在软体动物中获得的第一个Toll样受体的接头分子－CfMyd88基因，该结果直接证明软体动物具有与哺乳动物和节肢动物高度类似Myd88依赖的Toll样受体信号通路。同时通过这些基因组织分布的研究以及细菌结构LPS和PGN对这条通路上基因表达的影响，证明扇贝Toll信号通路可能与在果蝇中一样，参与扇贝的发育和免疫防御等多种功能。

Bromophenols coupled with nucleoside bases and brominated tetrahydroisoquinolines from the red alga Rhodomela confervoides

Three new bromophenols C-N coupled with nucleoside base derivatives (1-3) and three new brominated 1,2,3,4-tetrahydroisoquinolines (5-7, together with a new brominated tyrosine derivative (4, have been isolated from polar fractions of an ethanolic extract of the red alga Rhodomela confervoides. By spectroscopic and chemical methods including HRMS and 2D NMR data, their structures were determined as 7-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyl]-3,7-dihydro-1H-purine-2,6-dione (1), 7-(2,3-dibromo-4,5-dihydroxybenzyl)-3,7-dihydro-1H-purine-2,6-dione (2, 9-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyl]adenine (3), (-)-8S-(3-bromo-5-hydroxy-4-methoxy)phenylalanine (4), (-)-3S-8-bromo-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5), methyl (-)-3S-8-bromo-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (6), and methyl (-)-3S-6-bromo-8-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (7). Compounds 5-7 were semisynthesized by using 4 as the starting material.

青海歧穗大黄及其生长土壤中9种无机元素含量特征

为对比青海高原境内不同生境歧穗大黄中9种无机元素含量特征,用原子吸收光谱法分析了其宏量元素K、Na、Ca、Mg、P及微量元素Cu、Zn、Fe、Mn含量.结果表明,青海歧穗大黄植物与其土壤环境呈现出不同的无机元素含量特征,但一致表现为Ca和P在植物的富集,且植物中P含量随海拔升高而明显增加,Zn含量随海拔升高而增加.

荧光衍生试剂1-[2-（对甲苯磺酸醋）乙基]2-苯基咪唑[4,5-f]9,10-菲的合成及其在长链脂肪酸分析中的应用

合成了新的荧光衍生试剂1-[2-（对甲苯磺酸酯）乙基]-2-苯基咪唑[4,5-f]9,10-菲（TsEPIP），并将其作为柱前衍生化试剂，在Eclipse XDB-C：色谱柱上采用梯度洗脱实现了11种长链（C_(20)～C_(30)）游离脂肪酸（FFA）衍生物的基线分离。利用柱后在线的串联质谱并以大气压化学电离源（APCI）的正离子模式实现了各组分的质谱定性。对土壤及3种苔醉（东亚毛灰鲜、锦丝鲜、羽平鲜）中FFA组分的定量结果表明，苔鲜植物从土壤中富集了大量的长链游离脂肪酸。荧光检测的激发波长和发射波长分别为260 nm和380 nm。线性回归系数大于0.9996，检测限为26.19～76.67 fmol。所建立的方法具有良好的重现性，对实际样品的测定结果令人满意。

9种“藏茵陈”原植物中的7种有效化学成分研究

目的 研究藏药市场上9种“藏茵陈”植物中有效化学成分，根据所含成分的差异及其生理活性，确定这些植物的药用价值。方法 采用HPLC法测定7种成分的量。结果 9种植物的有效成分差异很大。结论 针对不同的化学成分用途，选择不同的植物，有效利用资源。[著者文摘]

青海青甘韭9个居群的核型

本文研究了青海葱属青甘韭 9个居群的染色体数目和核型。其中分布于湟源、西宁和共和海拔相对较低的地区的居群为 2倍体 ,核型公式为 2n =2x =16 =14m +2st (2SAT) (湟源居群和西宁居群 ) ,2n =2x =16 =12m +2sm +2st (2SAT) (共和居群 ) ;分布于玛沁、玉树、囊谦等高海拔地区的居群为 4倍体 ,核型公式为 2n =4x =32 =2 8m +4st (2SAT) (玉树居群 1和囊谦居群 1) ,2n =4x =32 =2 4m +4sm +4st (玛沁居群和玉树居群 3)和 2n =4x =32 =2 6m +2sm+4st (玉树居群 2 ) ;囊谦一个生长在林下的居群为8倍体，2n =8x =64 =54m +2sm +8st.讨论了居群间的核型分化和倍性与分布的关系。

CE determination of 2-(9-carbazole)ethyl chloroformate-labeled oligopeptides

A pre-column derivatization method for sensitive determination of oligopeptides, using the tagging reagent 2-(9-carbazole)ethyl chloroformate (CEOC-Cl) followed by capillary electrophoresis (CE) with diode-array detection, has been developed. Maximum yield close to 100% were observed when a three to fourfold molar excess of reagent was used at pH 9.0-10.0. Excess reagent was extracted with n-hexane-ethyl acetate 9:1-10:1 (v/v); this enabled direct analysis using CE with no significant disturbance from the major fluorescent reagent degradation by-products. The effects on the results of buffer pH and of SDS and organic modifier concentrations were examined. Good baseline resolution in the separation of five CEOC-peptides was achieved with a 48.5-cm total length (effective length 40 cm) 50-mu m inner diameter capillary column.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.